Assembly of bispecific antibodies

ABSTRACT

Described herein are methods for the efficient production of a heteromultimeric protein, such as a bispecific antibody. Heteromultimeric proteins may be capable of specifically binding more than one target molecule or different epitopes on a single target molecule. The methods modulate parameters to improve assembly of the heteromultimeric proteins at higher yield and efficiency than otherwise possible. Also described are compositions comprising a hinge-containing polypeptide, such as a half-antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2012/059810 having an international filing date of Oct. 11, 2012,the entire contents of which are incorporated herein by reference, andwhich claims benefit under 35 U.S.C. §119 to U.S. ProvisionalApplication Nos. 61/545,863, filed on Oct. 11, 2011, 61/546,503, filedOct. 12, 2011, 61/560,704, filed on Nov. 16, 2011 and 61/676,837, filedJul. 27, 2012. The disclosure of each of the above-referencedProvisional Applications is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

This disclosure relates to compositions and improved methods ofassembling heteromultimeric proteins such as bispecific antibodies.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Apr. 11, 2014, is named P4764C1SeqList.txt, and is 770bytes in size.

BACKGROUND

Monoclonal antibodies of the IgG type contain two identicalantigen-binding arms and a constant domain (Fc). Antibodies with adiffering specificity in their binding arms usually do not occur innature and, therefore, have to be crafted with the help of chemicalengineering (e.g., chemical cross-linking, etc.), recombinant DNA and/orcell-fusion technology.

Bispecific antibodies can bind simultaneously two different antigens.This property enables the development of therapeutic strategies that arenot possible with conventional monoclonal antibodies. The large panel ofimaginative bispecific antibody formats that has been developed reflectsthe strong interest for these molecules. See Berg J, Lotscher E, SteimerK S, et al., “Bispecific antibodies that mediate killing of cellsinfected with human immunodeficiency virus of any strain,” Proc NatlAcad Sci USA (1991) 88(11): 4723-4727 and Fischer N and Leger O.,“Biospecific Antibodies: Molecules That Enable Novel TherapeuticStrategies,” Pathobiology (2007) 74:3-14.

Another class of multispecific molecules is recombinant fusion proteins.Recombinant fusion proteins consisting of the extracellular domain ofimmunoregulatory proteins and the constant (Fc) domain of immunoglobulin(Ig) represent a growing class of human therapeutics. Immunoadhesinscombine the binding region of a protein sequence, with a desiredspecificity, with the effector domain of an antibody. Immunoadhesinshave two important properties that are significant to their potential astherapeutic agents: the target specificity, and the pharmacokineticstability (half-life in vivo that is comparable to that of antibodies).Immunoadhesins can be used as antagonist to inhibit or block deleteriousinteractions or as agonist to mimic or enhance physiological responses.See Chamow S M, Zhang D Z, Tan X Y, et al., “A humanized, bispecificimmunoadhesin-antibody that retargets CD3+ effectors to killHIV-1-infected cells,” J Hematother 1995; 4(5): 439-446.

Other multispecific molecules have been discussed elsewhere. Examplesinclude but are not limited to: Fisher et al., Pathobiology (2007)74:3-14 (review of various bispecific formats); U.S. Pat. No. 6,660,843,issued Dec. 9, 2003 to Feige et al. (peptibodies); US Pat. Publ. No.2002-004587 published Jan. 10, 2002 (multispecific antibodies); U.S.Pat. No. 7,612,181 issued Nov. 3, 2009 to Wu et al. (Dual VariableDomain format); U.S. Pat. No. 6,534,628, Nord K et al., Prot Eng (1995)8:601-608, Nord K et al., Nat Biotech (1997) 15:772-777, and Grönwall etal., Biotechnol Appl Biochem. (2008) June; 50(Pt 2):97-112 (Affibodies);Martens et al., Clin Cancer Res (2006), 12: 6144-6152 and Jin et al.,Cancer Res (2008) 68(11):4360-4368 (one armed antibodies); Bostrom etal., Science (2009) 323:1610-1614 (Dual Action Fab, aka mixed valencyantibodies). Other formats are known to those skilled in the art.

The manufacturing of clinical grade material remains challenging forantibodies generally and especially for the multispecific moleculesdescribed above. As noted above, there are many paths to the productionof molecules with mixed binding arms, i.e., binding arms that are notidentical to each other. But each of these methods has its drawbacks.

Chemical cross-linking is labor intensive as the relevant species mayyet need to be purified from homodimers and other undesirableby-products. In addition, the chemical modification steps can alter theintegrity of the proteins thus leading to poor stability. Thus, thismethod is often inefficient and can lead to loss of antibody activity.

Cell-fusion technology (e.g., hybrid hybridomas) express two heavy andtwo light chains that assemble randomly leading to the generation of 10antibody combinations. The desired heteromultimeric antibodies are onlya small fraction of the antibodies thus produced. Purification of thedesired heteromultimeric proteins dramatically reduces production yieldsand increases manufacturing costs.

Recombinant DNA techniques have been used to generate variousheteromultimeric formats, e.g., single chain Fv, diabodies, etc., thatdo not comprise an Fc domain. A major drawback for this type of antibodymolecule is the lack of the Fc domain and thus the ability of theantibody to trigger an effector function (e.g., complement activation,Fc-receptor binding etc.). Thus, a bispecific antibody comprising afunctional Fc domain is desired.

Recombinant DNA techniques have also been used to generate ‘knob intohole’ bispecific antibodies. See US Patent Application 20030078385(Arathoon et al.—Genentech). One constraint of this strategy is that thelight chains of the two parent antibodies have to be identical toprevent mispairing and formation of undesired and/or inactive moleculeswhen expressed in the same cell.

In addition, one of the limiting events during annealing andpurification is the redox efficiency. Oxidized heterodimer typicallyonly makes up 70-80% of the protein after this step (BioAnalyzer andMS-TOF). The remaining 20-30% of antibody is dimeric and lacks acovalent linkage (SEC-LLS). This can be removed but significantlyimpacts overall yields. Thus, there remains a need to improve theoverall yield in antibody production, especially heterodimers. Describedherein are methods that can improve overall yield of bispecificantibodies, heterodimers and the like. These and other aspects andadvantages of the invention will be apparent from the description of theinvention provided herein.

SUMMARY OF THE INVENTION

Production of heteromultimeric proteins assembled from two or morehinge-containing polypeptides, e.g., multispecific antibodies from twoor more half-antibodies, using current techniques has drawbacksincluding the production of a mixture of products, reduced yield anddecreased/elimination of effector function among others. In addition,aggregation and precipitation often occur during the preparation of eachhinge-containing polypeptide and during the assembly or annealing of theheteromultimers. Aggregation and precipitation can greatly reduce theyield of the desired heteromultimer. Thus, it is desirable to produceheteromultimeric proteins more efficiently and at higher levels.

Disclosed herein are efficient production processes/methods foreconomical production of heteromultimeric proteins, e.g., multispecificantibodies, by using or modulating one or more of the followingincluding without limitation: a stabilizer, a solubilizer, a reducingcondition, selected pH and selected temperature, etc. The inventivemethods described herein decreased loss of protein to precipitationand/or aggregation and improved the overall yield of heteromultimericprotein production, such as the production of bispecific antibodies.

In one aspect there is provided a method of forming or producing aheteromultimeric protein, said method comprising:

-   -   a. Providing a first hinge-containing polypeptide at pH 4-9,        preferably 5-9, in the presence of a first solubilizer, wherein        the first hinge-containing polypeptide comprises a        heteromultimerization domain;    -   b. Providing a second hinge-containing polypeptide at pH 4-9,        preferably 5-9, in the presence of a second solubilizer, wherein        the second hinge-containing polypeptide comprises a        heteromultimerization domain;    -   c. Mixing the first and second hinge-containing polypeptides in        a reducing condition to form an assembly mixture; and    -   d. incubating the assembly mixture to form or produce a        heteromultimeric protein comprising the first and second        hinge-containing polypeptides, wherein the first        hinge-containing polypeptide interacts with the second        hinge-containing polypeptide at the heteromultimerization        domain.

In certain embodiments of this aspect, step a and/or step b is precededby the step of purifying the first and/or second hinge-containingpolypeptide. In certain particular embodiments, the first and/or secondhinge-containing polypeptide is purified by Protein A.

In another aspect there is provided a method of forming or producing abispecific antibody, said method comprising:

-   -   a. Providing a first half-antibody at pH 4-9, preferably 5-9, in        the presence of a first solubilizer, wherein the first        half-antibody comprises a heteromultimerization domain;    -   b. Providing a second half-antibody at pH 4-9, preferably 5-9 in        the presence of a second solubilizer, wherein the second        half-antibody comprises a heteromultimerization domain;    -   c. Mixing the first and second half-antibodies in a reducing        condition to form an assembly mixture; and    -   d. incubating the assembly mixture to form or produce a        bispecific antibody comprising the first and second        half-antibodies, wherein the first half-antibody interacts with        the second half-antibody at the heteromultimerization domain.

In certain embodiments of this aspect, step a and/or step b is precededby the step of purifying the first and/or second half-antibody. Incertain particular embodiments, the first and/or second half-antibody ispurified by Protein A.

In a further aspect there is provided a method of producing aheteromultimer, said method comprising providing an arginine containingmixture of hinge-containing polypeptides said mixture having a pH ofbetween 4 and 9, preferably 5-9, adding a weak reductant and incubatingunder conditions so as to produce a heteromultimer.

In yet another aspect, there is provided a method of producing aheteromultimeric protein, said method comprising:

-   -   a. Obtaining a protein A purified first hinge-containing        polypeptide;    -   b. Obtaining a protein A purified second hinge-containing        polypeptide;    -   c. Adjusting the pH of each half-antibody to between 4 and 9;    -   d. Mixing the first and second hinge-containing polypeptide to        obtain an assembly mixture,    -   e. Adding a molar excess of a weak reductant to the assembly        mixture; and    -   f. incubating the assembly mixture to form a heteromultimeric        protein comprising the first and second hinge-containing        polypeptide.

In another aspect, there is provided a method of producing aheteromultimeric protein, said method comprising:

-   -   a. Obtaining a protein A purified first hinge-containing        polypeptide;    -   b. Obtaining a protein A purified second hinge-containing        polypeptide;    -   c. Adjusting the pH of each hinge-containing polypeptide to        between 4 and 9 in the presence of L-Arginine;    -   d. Mixing the first and second hinge-containing polypeptide to        obtain a mixed hinge-containing polypeptide pool, and    -   e. incubating to form a heteromultimeric protein comprising the        first and second hinge-containing polypeptide.

In certain embodiments of this aspect, the mixed hinge-containingpolypeptide pool is incubated in a reducing condition. In certainembodiments, the hinge-containing polypeptide comprises a half-antibody,an immunoadhesin or a functional fragment thereof. In certain otherembodiments, the Arginine is present at a concentration of 20 mM-1M, 20mM to 200 mM, or 50 mM-200 mM. In certain other embodiments, PVP isadded to the step d or step e. In certain embodiments, the pH isadjusted after mixing.

The instant applicants unexpectedly discovered that an intermediate pHhold of a hinge-containing polypeptide such as a half-antibody canpromote conformation shift that enhanced subsequent assembly of thehinge-containing polypeptides. In certain embodiments, the intermediatepH is between pH 4 and 9, preferably 5 and 9, or at least pH 5, at leastpH 5.5, at least pH 5.7, greater than pH 5, greater than pH 5.5, greaterthan pH 5.7, between 5 and 9, 5 and 8, 5.5 and 8, 5.5 and 9, 5.7 and 8,5.7 and 9, 6 and 8, 6 and 9, 7 and 8, 7.5 and 8.5, or 7 and 8.5. Asolubilizer can be added to prevent or minimize pH-induced precipitationof the hinge-containing polypeptide. In certain particular embodiments,the solubilizer is added before the intermediate pH hold. In certainembodiments, the first solubilizer and second solubilizer is eachselected from the group consisting of arginine, histidine and sucrose,preferably arginine and/or histidine. In certain other embodiments, thearginine is an arginine salt and/or histidine is a histidine salt. Incertain other embodiments, the arginine is an arginine derivative and/orhistidine is a histidine derivative. In certain other embodiments, thearginine or histidine is L-arginine or L-histidine. In certain otherembodiments, the arginine or histidine is arginine HCl or histidine HCl.In certain other embodiments, the arginine or histidine is argininephosphate or histidine phosphate. In certain embodiments, the first andsecond solubilizers are different; while in other embodiments, the firstand second solubilizers are the same. In certain preferred embodiments,both the first solubilizer and the second solubilizer comprise arginine.In yet other embodiments, the arginine is present at a concentration ofbetween 20 mM to 1M, 20 mM to less than 1M, 20 mM to 100 mM, 20 mM to200 mM, 20 mM to 300 mM, 20 mM to 400 mM, 50 mM to 100 mM, 50 mM to 150mM, 50 mM to 200 mM, 50 mM to 250 mM, or 50 mM to 300 mm, preferably 20mM to 200 mM. In yet other embodiments, the solubilizer comprises anarginine derivative including without limitation acetyl-arginine. Inother embodiments, both the first solubilizer and second solubilizercomprise histidine present at a concentration of between 20 mM to 1M, 20mM to less than 1M, 20 mM to less than 500 mM, 20 mM to 100 mM, 20 mM to200 mM, 20 mM to 300 mM, 20 mM to 400 mM, 50 mM to 100 mM, 50 mM to 150mM, 50 mM to 200 mM, 50 mM to 250 mM, 50 mM to 300 mm, 50 mM to 400 mM,50 mM to 500 mM, or 50 mM to 600 mM. In certain preferred embodiments,the solubilizer is added at a concentration of 50 mM. In certain otherembodiments, the solubilizer is added at a concentration of 200 mM. Incertain particular embodiments, arginine or histidine is added at aconcentration of 20 mM, 50 mM, 100 mM, or 200 mM. In certain otherparticular embodiments, the first and/or second hinge-containingpolypeptides are provided in the presence of both arginine andhistidine. In other embodiments, the arginine and histidine are eachpresent at a concentration of 20 mM to 1M, 20 mM to less than 1M, 20 mMto 100 mM, 20 mM to 200 mM, 20 mM to 300 mM, 20 mM to 400 mM, 50 mM to100 mM, 50 mM to 150 mM, 50 mM to 200 mM, 50 mM to 250 mM, or 50 mM to300 mM, preferably 50 mM to 200 mM.

In certain embodiments, the first and second hinge-containingpolypeptides are mixed before the intermediate pH hold (i.e, pHadjustment). In certain other embodiments, the first and secondhinge-containing polypeptides are mixed after the pH is adjusted in thefirst hinge-containing polypeptide and second hinge-containingpolypeptide separately. In certain embodiments, a solubilizer is addedbefore pH adjustment.

In certain embodiments, the first hinge-containing polypeptide and thesecond hinge-containing polypeptide are separately purified beforemixing; while in other embodiments, the first hinge-containingpolypeptide and the second hinge-containing polypeptide are co-purifiedafter mixing. In certain particular embodiments, the hinge-containingpolypeptide comprises a half-antibody. In certain embodiments, theassembled heteromultimeric protein can be subjected to furtherpurification.

Any suitable methods can be used for purification including withoutlimitation purification by protein A chromatography, protein Gchromatography, hydrophobic interaction chromatography (HIC),fractionation on immunoaffinity column, ethanol precipitation, reversephase chromatography on silica or on an ion-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75, and other similarpurification methods, and combinations thereof.

In certain embodiments, the hinge-containing polypeptide such as ahalf-antibody is purified by Protein A or Protein G chromatography. Inanother embodiment the first and second hinge-containing polypeptidesare mixed prior to Protein A purification and co-purified over ProteinA. In certain embodiments, the pH is adjusted after mixing the Protein Apurified polypeptides. In other embodiments, the pH is adjusted prior tomixing the Protein A purified polypeptides. In certain embodiments, asolubilizer is added before pH adjustment.

In certain other embodiments, the hinge-containing polypeptide ispurified by HIC or an ion-exchange column. It is within the ability of aperson skilled in the art to select suitable purification methods. Forexample, the hinge-containing polypeptide can be purified by a Protein Acolumn followed by an ion-exchange column; the hinge-containingpolypeptide can also be purified by a Protein A column followed by a gelfiltration column and/or HIC. In other examples, the hinge-containingpolypeptide can be purified by one or more ion-exchange column beforepurification by a Protein A column. In certain embodiments, the washingand/or elution buffers used during any of the purification steps of thehinge-containing polypeptides do not contain arginine and/or histidine.

In yet other embodiments, the half-antibody eluted from the Protein Amatrix or other column matrix at acidic pH is adjusted to anintermediate pH. This subsequent pH adjustment (also referred to as anintermediate pH hold) can cause precipitation of the hinge-containingpolypeptide such as a half-antibody and thus lead to reduced yield ofthe assembled heteromultimeric protein. Thus, in certain embodiments,the half-antibody eluted from the Protein A or Protein G column atacidic pH is provided in the presence of a solubilizer before pHadjustment. In the event that a pH adjustment step is not necessary, incertain embodiments a solubilizer is preferably added to the purifiedhinge-containing polypeptide to prevent or reduce precipitation and/oraggregation.

In addition to intermediate pH hold, the instant applicants unexpectedlydiscovered that heating can enhance conformation shift and/or assemblyof the hinge-containing polypeptides such as half-antibodies.Accordingly, in certain embodiments, one, more or all of the steps a-dof the inventive methods are heated at a temperature of between 15° C.and 39° C., 15° C. and 42° C., 18° C. and 37° C., 20° C. and 42° C., 20°C. and 25° C., 25° C. and 42° C., 25° C. and 35° C., 25° C. and 39° C.,30° C. and 35° C., 32° C. and 35° C. or 32° C. and 37° C., preferably35° C. and 37° C., for at least 30 minutes. In certain embodiments, theincubation time is up to 72 hours, especially at room temperature. Insome embodiments the incubation time is 3 hours at 35° C. In certainother embodiments, the temperature is at or about 30° C., 35° C., or 37°C.

Heating, however, can also increase aggregation and/or precipitation.Accordingly, in certain particular embodiments, a solubilizer is addedto the half-antibody eluted from a Protein A or Protein G column beforeheating.

In certain embodiments, the hinge-containing polypeptide comprises ahalf-antibody, an immunoadhesin, or a functional fragment thereof. Incertain particular embodiments, the hinge-containing polypeptidecomprises an Fc component.

In certain particular embodiments, the first and/or secondhinge-containing polypeptide comprises a half-antibody. In certainembodiments, the half-antibody is an IgG half-antibody. In certainparticular embodiments, the IgG half-antibody is of the IgG1, IgG4 orIgG2 isotype. In certain advantageous embodiments, the signal peptide ofthe immunoglobulin molecule is retained to facilitate secretion of thehalf-antibody especially when produced in mammalian cells. In certainembodiments, the inventive method comprises providing a first and asecond half-antibody, at pH 5-9 in the presence of arginine at aconcentration of about 50 mM, and alternatively or additionallyhistidine at a concentration of about 200 mM. In certain embodiments,the first and/or second half-antibody each comprises an antigen bindingdomain specific for a different antigen or a different epitope on thesame antigen and the assembled full antibody is a bispecific antibody.In certain other embodiments, the first and second half-antibodies areof the same isotype; while in other embodiments, the first and secondhalf-antibody are of different isotypes.

The half-antibody may comprise a V_(L) domain, a V_(H) domain, a hingedomain, a CH₂ domain and/or a CH₃ domain. The half-antibody can also bea single chain polypeptide further comprising a tether, wherein saidsingle chain polypeptide comprises domains positioned relative to eachother in an N-terminal to C-terminal direction as follows:V_(L)-tether-V_(H)-hinge-CH₂—CH₃. In certain other embodiments, thehalf-antibody further comprises a C_(L) domain and a CH₁ domain; and infurther embodiments, the half-antibody can be a single chain polypeptidefurther comprising a tether, wherein said single chain polypeptidecomprises domains positioned relative to each other in an N-terminal toC-terminal direction as follows:V_(L)-C_(L)-tether-V_(H)-CH₁-hinge-CH₂—CH₃.

The tether may comprise one or more Glycine (G) and Serine (S) residues.In other embodiments, the tether comprises GGS repeats. The tether, forexample, is between 15 and 50 amino acids in length. In a particularembodiment, the tether is between 20 and 32 amino acids in length, forexample, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 aminoacids in length. In certain embodiments, the tether is cleavable. Inother embodiments, the tether may or may not be cleavable from theprotein. In certain preferred embodiments, the tether is cleavable intwo sites at or near the N and C terminus of the tether by the sameenzyme. In one embodiment, the tether comprises the cleavage site forproteases such as furin. In a further embodiment, the tether is cleavedby furin at the cleavage site RXRXRR (SEQ ID NO:1), wherein X is anyamino acid. In some embodiments, the first hinge-containing polypeptideis a half-antibody and second hinge-containing polypeptide is singlechain half-antibody.

In another embodiment, the invention provides a protein comprising atether and an Fc component complex, wherein the tether may or may not becleavable from the protein.

In further embodiments, the first and second hinge-containingpolypeptides comprise a heteromultimerization domain. Theheteromultimerization domain may be a knob into hole mutation, leucinezippers, electrostatic, and the like. The first hinge-containingpolypeptide may comprise a knob and the second hinge-containingpolypeptide may comprise a hole. In certain embodiments, thehinge-containing polypeptide comprises a half-antibody, and the firsthalf-antibody comprises a knob and the second half-antibody comprises ahole.

In some embodiments, the methods comprises adding Arginine to a finalconcentration of between 20 mM to 1M prior to adjusting the pH. In someembodiments the Arginine is added to a final concentration of between 50mM-600 mM. In some embodiments the Arginine is added to a finalconcentration of between 50 mM-100 mM.

In certain embodiments, the method includes incubating eachhinge-containing polypeptide Protein A pool at a pH of between 5 and 8prior to mixing the first and second half-antibodies. In otherembodiments the Protein A pools are mixed and then the pH adjusted tobetween 5 and 8.

In some embodiments, the methods described herein comprise incubatingthe mixed half-antibody or mixed hinge-containing polypeptide pool at atemperature of between 15° C. and 39° C., preferably between 18° C.-37°C., more preferably between 20° C.-25° C., more preferably between 32°C.-37° C., for at least 30 minutes.

The hinge-containing polypeptides can be produced by for example, abacterial cell, a yeast cell, a baculovirus in an insect cell, or amammalian cell. In certain embodiments, the hinge-containing polypeptideis produced by a bacteria cell, particularly E. coli. In certain otherparticular embodiments, the hinge-containing polypeptide is produced bya mammalian cell, particularly a CHO cell. In certain particularembodiments, the hinge-containing polypeptide comprises a half-antibody.

The hinge-containing polypeptides can interact to form a dimer ormultimer via the heterodimerization domain. In certain embodiments, theinteraction between the first and second hinge-containing polypeptidesat the interface of the heterodimerization domains is aprotuberance-into-cavity interaction, a hydrophobic interaction and/oran electrostatic interaction. In certain other embodiments, theheteromultimerization domain comprises a knob (e.g., protuberance), ahole (e.g., cavity), a leucine zipper, a coiled coil, or a polar aminoacid residue capable of forming an electrostatic interaction, orcombinations thereof. In certain embodiments, the first hinge-containingpolypeptide comprises a knob, and the second hinge-containingpolypeptide comprises a hole. In certain other embodiments, theinteraction involves both a hydrophobic interaction and an electrostaticinteraction. In certain exemplary embodiments, the heteromultimerizationdomain of each of the first and second hinge-containing polypeptidescomprises either a knob or a hole and an amino acid residue capable offorming an electrostatic interaction. It is understood by one skilled inthe art that the heterodimerization domain can comprise more than oneway of interaction, for example, knob and hole (K&H) and hydrophobicinteraction, K&H and leucine zipper, etc. In certain embodiments, thehinge-containing polypeptide further comprises a tether. In certainparticular embodiments, the hinge-containing polypeptide comprises ahalf-antibody.

In certain particular embodiments, the assembly mixture is present in areducing condition, preferably a weak reducing condition, having anoxidation potential of between −50 to −600 mV, −100 to −600 mV, −200 to−600 mV, −100 to −500 mV, −150 to −300 mV, more preferably between −300to −500 mV, most preferably about −400 mV, under conditions that promotethe assembly of the heteromultimeric proteins such as when the pH isbetween 7 and 9, and the temperature is between 15° C. and 39° C. Incertain embodiments, a reductant is added to step c or step d to preparea desired reducing condition during assembly. In certain otherembodiments, the reductant is selected from the group consisting ofdithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), thioglycolicacid, ascorbic acid, thiol acetic acid, glutathione (GSH),Beta-MercaptoEthylAmine, cysteine/cystine, glutathione (GSH),cysteamine/cystamine, glycylcysteine, and beta-mercaptoethanol,preferably GSH. In certain preferred embodiments, the reductant,preferably a weak reductant, is selected from the group consisting ofglutathione (GSH), Beta-MercaptoEthylAmine, cysteine/cystine,glutathione (GSH)/glutathione disulfide (GSSG), cysteamine/cystamine,glycylcysteine, and beta-mercaptoethanol, and preferably GSH. In certainembodiments, the reductant is not DTT.

In certain other embodiments, the reductant is added to the assemblymixture in 2-600×, 2-200×, 2-300×, 2-400×, 2-500×, 2-20×, 2-8×, 20-50×,50-600×, 50-200×, or 100-300× molar excess, preferably 50-400×, morepreferably 100-300×, and most preferably 200×, molar excess with respectto the total amount of the hinge-containing polypeptides. In certainembodiments, the assembly mixture has a pH of between 7 and 9,preferably pH 8.5. In certain embodiments, the hinge-containingpolypeptide is a half-antibody.

In some embodiments, the reductant is added to the first and secondhinge-containing polypeptides prior to mixing. Preferably the additionis less than 1 hour, more preferably less than 15 minutes, mostpreferably less than 5 minutes, before mixing.

In certain embodiments, the method further comprises adding a stabilizerto the reaction in one or more of the steps, including withoutlimitation the intermediate pH hold step and the assembly step in thepresence or absence of heating. For example, a stabilizer can be addedto the hinge-containing polypeptide to prevent or reduce aggregation. Inother examples, a stabilizer can be added to the assembly mixture toprevent or reduce aggregation during the assembly of a heteromultimericprotein. In certain particular embodiments, the hinge-containingpolypeptide comprises a half-antibody.

In certain particular embodiments, the stabilizer is selected from thegroup consisting of arginine, histidine and Polyvinylpyrrolidone (PVP).In certain embodiments, the arginine or histidine is an arginine salt orhistidine salt. In certain other embodiments, the arginine or histidineis an arginine derivative or histidine derivative. In certain otherembodiments, the arginine or histidine is arginine HCl or histidine HCl.In certain embodiments, the arginine is not arginine phosphate.

In certain other embodiments, the method further comprises the step ofincubating the assembly mixture in the presence of PVP. In relatedembodiments, the PVP is added up to 40% (w/v). In certain otherembodiments, the PVP is present in the assembly mixture is at aconcentration of 2%-6% (w/v), 10%-20%, 2%-10%, 1%, 1.3%, 1.7%, 2%, 2.3%,2.7%, 3%, 3.3%, 3.7% or 4%, preferably 0.1%-10%, more preferably 2%-6%,and most preferably 4%. In certain embodiments, the PVP is no more than100 KD, no more than 30 KD, and preferably 10 KD. In certain otherembodiments, the PVP is present in less than 10% (w/v), or less than 5%(w/v).

In some embodiments, the stabilizer is arginine present at aconcentration of between 20 mM to 1M, 20 mM to less than 1M, 20 mM to100 mM, 20 mM to 200 mM, 20 mM to 300 mM, 20 mM to 400 mM, 20 mM to 50mM, 50 mM to 100 mM, 50 nM to 150 mM, 50 mM to 200 mM, 50 mM to 250 mM,or 50 mM to 300 mM. In other embodiments, the stabilizer is histidinepresent at a concentration of between 20 mM to 1M, 20 mM to less than1M, 20 mM to 100 mM, 20 mM to 200 mM, 20 mM to 300 mM, 20 mM to 400 mM,20 mM to 50 mM, 50 mM to 100 mM, 50 nM to 150 mM, 50 mM to 200 mM, 50 mMto 250 mM, or 50 mM to 300 mM. In certain preferred embodiments, thearginine or histidine is added at a concentration of 50 mM or 200 mM. Incertain other embodiments, the arginine and/or histidine is added at aconcentration of 20 mM to 200 mM, 20 mM to 100 mM, 50 mM to 200 mM or 50mM to 100 mM. In certain other embodiments, the hinge-containingpolypeptide comprises a half-antibody.

In yet another aspect, the invention provides a host cell expressing ahinge-containing polypeptide. In certain embodiments, thehinge-containing polypeptide comprises a half-antibody.

In a further aspect, the invention provides a method of producing abispecific antibody, comprising the steps of (a) culturing a first hostcell engineered to express a first half-antibody specific for a firstantigen or a first epitope of an antigen; (b) culturing a second hostcell engineered to express a second half-antibody specific for a secondantigen or a second epitope of the same antigen; (c) obtaining the firsthalf-antibody from the culture of step a at pH between 4-9, preferably5-9, in the presence of a first solubilizer; (d) obtaining the secondhalf-antibody from the culture of step b at pH between 4-9, preferably5-9, in the presence of a second solubilizer; (e) mixing the first andsecond half-antibodies in a reducing condition to form an assemblymixture; and (f) incubating the assembly mixture to form a bispecificantibody comprising the first and second half-antibodies.

In some embodiments, the first host cell and the second host cell arecultured in separate cultures, and the first half-antibody and thesecond half-antibody are separately purified from the cultures of thefirst and second host cells before mixing. In certain embodiments, thefirst and second host cells are cultured in separate cultures, thecultures are combined, the cells pelleted, optionally homogenized and/orlysed, and the first and second half-antibodies are co-purified by anysuitable methods. In certain embodiments, the first and secondhalf-antibodies are co-purified by protein A purification. In furtherembodiments, the first and second host cells are co-cultured in a mixedculture and the first and second half-antibodies are co-purified.

The host cell can be, for example, a bacterial cell, a yeast cell, aplant cell, an insect cell or a mammalian cell. In certain particularembodiments, the hinge-containing polypeptide or half-antibody isproduced by a mammalian cell, such as a CHO cell. In certain otherembodiments, the host cell is a bacteria cell, in particular E. coli.

In certain additional embodiments, the inventive methods furthercomprise the step of recovering the heteromultimeric protein orbispecific antibody formed in step (d). The assembled heteromultimericprotein can be further purified by methods described throughout theapplication or suitable methods known in the art.

In a further aspect, the invention provides compositions comprising ahinge-containing polypeptide and a solubilizer, wherein the pH of thecomposition is between pH 4-pH 9, preferably between pH 5-9. In certainembodiments, the pH of the composition is at least pH 5, at least pH5.5, at least pH 5.7, greater than pH 5, greater than pH 5.5, greaterthan pH 5.7, between 5 and 9, 5 and 8, 5.5 and 8, 5.5 and 9, 5.7 and 8,5.7 and 9, 6 and 8, 6 and 9, 7 and 8, 7.5 and 8.5, or 7 and 8.5. Incertain embodiments, the solubilizer is selected from the groupconsisting of arginine, histidine and sucrose, preferably arginineand/or histidine. In certain other embodiments, the arginine is anarginine salt and/or histidine is a histidine salt. In certain otherembodiments, the arginine is an arginine derivative and/or histidine isa histidine derivative. In certain other embodiments, the arginine orhistidine is L-arginine or L-histidine. In certain other embodiments,the arginine or histidine is arginine HCl or histidine HCl.

In certain particular embodiments, the solubilizer is arginine orhistidine. In certain other embodiments, the arginine or histidine ispresent at a concentration of 20 mM, 50 mM, 200 mM, 400 mM, between 20mM to 1M, between 20 mM to less than 1M, 20 mM and 200 mM, 20 mM to 400mM, 20 mM to 100 mM, 50 mM to 100 mM, 50 mM to 200 mM, 50 mM to 300 mMor 50 mM to 400 mM. In certain particular embodiments, the compositioncomprising both arginine and histidine each present at a concentrationof 20 mM, 50 mM, 200 mM, 400 mM, between 20 mM to 1M, between 20 mM toless than 1M, 20 mM and 200 mM, 20 mM to 400 mM, 20 mM to 100 mM, 50 mMto 100 mM, 50 mM to 200 mM, 50 mM to 300 mM or 50 mM to 400 mM. Incertain other embodiments, the composition does not comprise guanidineHCl or urea. In certain embodiments, the composition alternatively oradditionally comprises a stabilizer.

In certain particular embodiments, the hinge-containing polypeptidecomprises a half-antibody. In certain other particular embodiments, thecomposition comprises only one type of hinge-containing polypeptide orhalf-antibody. In certain embodiments, the composition comprises onlyone type of half-antibody that is a knob half-antibody. In certain otherembodiments, the composition comprises only one type of half-antibodythat is a hole half-antibody.

In certain particular embodiments, the composition further comprises asecond hinge-containing polypeptide, wherein the first hinge-containingpolypeptide comprises a knob and the second hinge-containing polypeptidecomprises a hole. In certain embodiments, the hinge-containingpolypeptide comprises a half-antibody. In certain particularembodiments, the hinge-containing polypeptide is a half-antibody. Incertain other embodiments, the half-antibody is of the IgG1, IgG2 orIgG4 isotype.

All embodiments disclosed herein can be combined unless the contextclearly dictates otherwise. In addition, any and every embodimentdescribed above applies to any and every aspect of the invention, unlessthe context clearly indicates otherwise.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a fully oxidized half-antibody. Not shown is the“knob” or “hole” or other heterodimerization domains. The half-antibodydepicted in this figure is an IgG1 isotype. One skilled in the art willappreciate that the other immunoglobulin isotypes can be envisioned ashalf-antibodies with the corresponding inter- and intra-chain bonds. Inan intact antibody the hinge cysteines will form inter-chain disulfidebonds.

FIG. 1B illustrates a full-length bispecific antibody with aheteromultimerization domain. Not depicted are the inter-heavy chaindisulfide bonds in the hinge region. The heteromultimerization domainshown is the knob into hole format.

FIG. 1C is a cartoon representation of a bispecific antibody comprisinga heteromultimerization domain (knob into hole), a furin cleavabletether and an optional extra disulfide bond (S354). The inter-heavychain disulfide bonds in the hinge region are also shown. The furincleavage sites are indicated by the triangles. Although the furincleavable tether is shown on the half-antibody comprising the knob itcan also be utilized on the hole half-antibody or on both the knob andhole half-antibodies.

FIGS. 2A-B show composite size exclusion chromatograms demonstrating theeffects of pH on the conformation shift of half-antibodies. FIG. 2Ashows that elevated pH induced hole half-antibody conformation shiftresulting in larger hydrodynamic radius. Such a conformation shiftenhanced heterodimerization during assembly. FIG. 2B shows that elevatedpH promoted formation of non-covalent knob half-antibody homodimer. Sucha conformation shift favored bispecific formation during assembly.Reference is made to Example 2.

FIGS. 3A-3B depict the results showing that a solubilizer such asarginine (FIGS. 3A and B) or histidine hydrochloride (FIG. 3B) reducedintermediate pH-induced precipitation of knob half-antibodies.

FIGS. 4A-B show composite chromatograms demonstrating the effect of areductant such as glutathione on aggregation and bispecific antibodyassembly. Glutathione is added in 2-200× molar excess. Reference is madeto Example 3.

FIG. 5A is a graph illustrating the effect of temperature on the rate ofIgG1 bispecific antibody formation (assembly). FIG. 5B shows thatincreased temperature promoted assembly of knob-into-hole bispecificIgG1 antibody in the presence of 200× molar excess of glutathione withor without pH hold as analyzed by reverse phase chromatography. FIG. 5Balso illustrates that optimization of pH hold of the half antibody poolsto drive conformation shifts prior to assembly improved the rate andefficiency of assembly. Reference is made to Example 4.

FIG. 6A illustrates the effect of a stabilizer such as PVP onstabilizing the formed bispecific antibody and reduction in aggregateformation.

FIG. 6B shows that PVP and histidine at elevated temperature promotedassembly of knob-into-hole bispecific IgG4 antibody as analyzed byreverse phase chromatography. Bottom curve: room temperature assemblywith 300× Glutathione:Ab ratio, pH=8.5, ˜400 mM Arginine; top curve:heated assembly with 200× Glutathione:Ab ratio, pH=8.0, 4% PVP, 50 mMArginine, 100 mM Histidine at 35° C.

FIG. 6C demonstrates that PVP reduced aggregation of IgG4 knob-into-holebispecific antibody during heated assembly at 37° C. and pH 8 for 6hours. Reference is made to Example 5.

FIG. 7 illustrates that histidine reduced heat-induced aggregation ofIgG4 hole half-antibodies at 37° C. and pH 8 for 3 hours.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxylorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxyl orientation, respectively.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to “a host cell” means one or more host cells.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

All embodiments disclosed herein can be combined unless the contextclearly dictates otherwise. In addition, any and every embodimentdescribed below applies to any and every aspect of the invention, unlessthe context clearly indicates otherwise.

I. Definitions

The instant invention provides methods of producing a heteromultimericprotein comprising a first hinge-containing polypeptide and a secondhinge-containing polypeptide. The term “hinge-containing polypeptide” asused herein refers to a polypeptide that contains at least one hingeregion. In certain embodiments, the hinge region connects multipledomains, for example, a binding domain and an effector domain, andprovides some structurally flexibility to the polypeptide fordimerization or multimerization. As an example, the binding domain canbe an antigen binding domain of an antibody or a ligand binding domainof a receptor, and the effector domain can be an Fc component of anantibody. In certain embodiments, the first hinge-containing polypeptideis different from the second hinge-containing polypeptide, and theresulting dimer or multimer is a heterodimer or heteromultimer. Incertain particular embodiments, the first hinge-containing polypeptideand the second hinge-containing polypeptide bind to two differentepitopes on the same target protein. In certain other embodiments, thefirst hinge-containing polypeptide has a different target bindingspecificity from that of the second hinge-containing polypeptide and theresulting heterodimer or heteromultimer binds to two or more differenttarget proteins. In certain embodiments, the hinge-containingpolypeptide comprises either a naturally occurring or engineeredheterodimerization domain. In certain particular embodiments, thehinge-containing polypeptide comprises one or more cysteine residues inthe hinge region capable of forming one or more di-sulfide bonds withanother hinge-containing polypeptide.

A hinge-containing polypeptide includes without limitation ahalf-antibody, an immunoadhesin, and functional fragment thereof. Theterm “functional fragment” as used herein refers to a fragment, i.e.,less than the full-length, of the hinge-containing polypeptide, whichstill retains the desired function, for example, retaining the target orantigen-binding activity, the Fc effector activity and/ordimerization/multimerization ability. In certain particular embodiments,the first hinge-containing polypeptide and second hinge-containingpolypeptide each is a half-antibody with different antigen bindingspecificity, and the resulting dimer or multimer is a bispecific ormultispecific antibody. In certain embodiments, the resultingheteromultimeric protein comprises a half-antibody and an immunoadhesin.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody that has polyepitopic specificity. Suchmultispecific antibodies include, but are not limited to, an antibodycomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), where the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains witheach V_(H)V_(L) unit binding to a different epitope, antibodies havingtwo or more single variable domains with each single variable domainbinding to a different epitope, full length antibodies, antibodyfragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodiesand triabodies, antibody fragments that have been linked covalently ornon-covalently. “Polyepitopic specificity” refers to the ability tospecifically bind to two or more different epitopes on the same ordifferent target(s). “Monospecific” refers to the ability to bind onlyone epitope. According to one embodiment the multispecific antibody isan IgG antibody that binds to each epitope with an affinity of 5 μM to0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1μM to 0.001 pM.

A naturally occurring basic 4-chain antibody unit is a heterotetramericglycoprotein composed of two identical light (L) chains and twoidentical heavy (H) chains (an IgM antibody consists of 5 of the basicheterotetramer units along with an additional polypeptide called Jchain, and therefore contains 10 antigen binding sites, while secretedIgA antibodies can polymerize to form polyvalent assemblages comprising2-5 of the basic 4-chain units along with J chain). In the case of IgGs,the 4-chain unit is generally about 150,000 daltons. Each L chain islinked to an H chain by one covalent disulfide bond, while the two Hchains are linked to each other by one or more disulfide bonds dependingon the H chain isotype. Each H and L chain also has regularly spacedintrachain disulfide bridges. Each H chain has, at the N-terminus, avariable domain (V_(H)) followed by three constant domains (C_(H)) foreach of the α and γ chains and four C_(H) domains for p and E isotypes.Each L chain has, at the N-terminus, a variable domain (V_(L)) followedby a constant domain (C_(L)) at the C-terminus. The V_(L) is alignedwith the V_(H) and the C_(L) is aligned with the first constant domainof the heavy chain (C_(H)1). Particular amino acid residues are believedto form an interface between the light chain and heavy chain variabledomains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thedition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting abeta-sheet configuration, connected by three hypervariable regions,which form loops connecting, and in some cases forming part of, thebeta-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework regions” (FR) are those variable domain residues other thanthe CDR residues. Each variable domain typically has four FRs identifiedas FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat,the light chain FR residues are positioned at about residues 1-23(LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavychain FR residues are positioned about at residues 1-30 (HCFR1), 36-49(HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues.If the CDRs comprise amino acid residues from hypervariable loops, thelight chain FR residues are positioned about at residues 1-25 (LCFR1),33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain andthe heavy chain FR residues are positioned about at residues 1-25(HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavychain residues. In some instances, when the CDR comprises amino acidsfrom both a CDR as defined by Kabat and those of a hypervariable loop,the FR residues will be adjusted accordingly. For example, when CDRH1includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat. In one embodiment, for the VL, the subgroup is subgroup kappaI as in Kabat. In one embodiment, for the VH, the subgroup is subgroupIII as in Kabat.

One example of an “intact” antibody is one that comprises anantigen-binding site as well as a C_(L) and at least heavy chainconstant domains, C_(H)1, C_(H)2, and C_(H)3. The constant domains canbe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variant thereof.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or a variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab)₂, andFv fragments; diabodies (Db); tandem diabodies (taDb), linear antibodies(e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10):1057-1062 (1995)); one-armed antibodies, single variable domainantibodies, minibodies, single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments (e.g., includingbut not limited to, Db-Fc, taDb-Fc, taDb-CH3 and (scFV)4-Fc).

The term “half-antibody” as used herein refers to one immunoglobulinheavy chain associated with one immunoglobulin light chain. An exemplaryhalf-antibody is depicted in FIG. 1A. One skilled in the art willreadily appreciate that a half-antibody may encompass a fragment thereofand may also have an antigen binding domain consisting of a singlevariable domain, e.g., originating from a camelidae.

The instant inventors unexpectedly discovered that pH optimization oradjustment of half-antibodies eluted from a Protein A column or othermatrix at low pH induced conformation shift of hinge-containingpolypeptides such as half-antibodies. The pH optimization to anintermediate pH, sometimes referred to as pH hold or intermediate pHhold throughout this disclosure, may cause precipitation or aggregationof the half-antibodies. Thus, in certain embodiments, the method ofproducing a heteromultimeric protein comprises the step of providing afirst or second hinge-containing polypeptide at pH 5-9 in the presenceof a first or a second solubilizer, respectively.

A solubilizer as used herein is defined as a reagent that prevents orreduces precipitation of a hinge-containing polypeptide, such as ahalf-antibody. Suitable solubilizer includes without limitation,arginine and histidine, or a salt or derivative thereof, and sucrose. Incertain embodiments, the solubilizer is arginine and/or histidine. Incertain embodiments, solubilizer prevents or reduces precipitationinduced by intermediate pH hold and/or heating. In certain particularembodiments, a solubilizer is added before the intermediate pH hold(i.e., before adjusting to intermediate pH), and/or heating. In certainembodiments, the arginine or histidine is an arginine salt or histidinesalt. In certain other embodiments, the arginine or histidine is anarginine derivative or histidine derivative. Reduction of precipitationcan lead to increased yield of the desired assembled final product.

Imidazole and guanidine have been used to solubilize protein for generalprotein preparation and purification. However, it was unexpectedlydiscovered that imidazole and guanidine alone, without being in thecontext of histidine or arginine, respectively, were insufficient toimprove the overall yield of assembled heteromulimeric protein asdescribed herein, such as bispecific antibody. In certain embodiments,imidazole and guanosine can denature the proteins.

Similarly, detergents such as guanidine HCl and urea are commonly usedto reduce aggregation/precipitation in general but they can completelydenature the protein. Thus, in certain embodiments, the solubilizerprevents or reduces precipitation without denaturing the protein ofinterest. Thus, in certain particular embodiments, the solubilizer isnot guanidine HCl, guanidine, imidazole or urea. And in certain otherembodiments, the compositions of the invention do not comprise guanidineHCl or urea. In certain other embodiments, the solubilzer is not Tweenor PEG.

In addition, a stabilizer can be added, for example, during anintermediate pH hold of each half-antibody or during assembly at orright after mixing the hinge-containing polypeptides or half-antibodies.A stabilizer can be added to the reaction of one or more or all of thesteps of the inventive methods to prevent or reduce aggregation of thehinge-containing polypeptides or half-antibodies, before, during and/orafter assembly.

Aggregates can be detected as high molecular weight species and, in thecontext of half-antibodies, high molecular weight species with amolecular weight larger than 150 kDa. Aggregates can be detected andquantified by, for example, size exclusion chromatography or othersuitable methods. In certain other embodiments, the aggregates detectedby the size exclusion chromatography can pass through a 0.2 um sterilefilter. Precipitated proteins, on the other hand, can be composed ofdenatured proteins or aggregated proteins which can form very largecomplex. Several reagents have been tested and were determinedineffective or not suitable for use as stabilizers, for example,imidazole, 3-(N-morpholino)propanesulfonic acid (MOPS),2-(N-morpholino)ethanesulfonic acid (MES), cyclodextrin, CuSO₄ andNaOAc. Thus, in certain embodiments, the stabilizer does not includeinorganic salts or transition metals. Suitable stabilizer includeswithout limitation PVP, histidine and arginine. Reduction of aggregationcan lead to increased yield of the desired assembled final product.

PVP is a water soluble uncharged polymer with a pyrrolidone group. Incertain embodiments, other uncharged polar polymers, other reagents orcompounds, especially compounds with similar structure and propertieswith the suitable stabilizers described herein may be suitablestabilizers for use in the invention. It is within the ability of oneskilled in the art to determine a suitable stabilizer by analyzing theeffect of the compound on the levels of aggregation by methods known inthe art, including the methods provided herein.

A reagent may be characterized as both a solubilizer and a stabilizer.For example, arginine can be used as a solubilizer to reduceprecipitation of half-antibodies during intermediate pH hold and/orheating, as well as a stabilizer to reduce aggregation during theassembly step. Similarly, histidine can be used as a solubilizer toreduce precipitation as well as a stabilizer to reduce aggregation ofhalf-antibodies, during the intermediate pH hold and/or heating. Withoutbeing limited to any particular mechanisms, in certain embodiments, botha solubilizer and a stabilizer can work by preventing interaction ofhydrophobic patches on the surfaces of proteins that can lead toaggregation. In other embodiments, both a solubilizer and a stabilizercan function by forming clathrates to prevent undesirable interaction ofproteins.

The term “single chain half-antibody” as used herein refers to a singlechain polypeptide comprising a VL domain, optionally a CL domain, atether, a VH domain, optionally a CH1 domain, a hinge domain, a CH2domain and a CH3 domain, wherein said domains are positioned relative toeach other in an N-terminal to C-terminal direction as follows:VL-tether-VH-hinge-CH2-CH3 or VL-CL-tether-VH-CH1-hinge-CH2-CH3.

The expression “single domain antibodies” (sdAbs) or “single variabledomain (SVD) antibodies” generally refers to antibodies in which asingle variable domain (VH or VL) can confer antigen binding. In otherwords, the single variable domain does not need to interact with anothervariable domain in order to recognize the target antigen. Examples ofsingle domain antibodies include those derived from camelids (lamas andcamels) and cartilaginous fish (e.g., nurse sharks) and those derivedfrom recombinant methods from humans and mouse antibodies (Nature (1989)341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001)26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO 02/051870).

The expression “linear antibodies” generally refers to the antibodiesdescribed in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The term “knob-into-hole” or “KnH” technology as mentioned herein refersto the technology directing the pairing of two polypeptides together invitro or in vivo by introducing a protuberance (knob) into onepolypeptide and a cavity (hole) into the other polypeptide at aninterface in which they interact. For example, KnHs have been introducedin the Fc:Fc binding interfaces, CL:CH1 interfaces or VH/VL interfacesof antibodies (e.g., US2007/0178552, WO 96/027011, WO 98/050431 and Zhuet al. (1997) Protein Science 6:781-788). This is especially useful indriving the pairing of two different heavy chains together during themanufacture of multispecific antibodies. For example, multispecificantibodies having KnH in their Fc regions can further comprise singlevariable domains linked to each Fc region, or further comprise differentheavy chain variable domains that pair with similar or different lightchain variable domains. KnH technology can also be used to pair twodifferent receptor extracellular domains together or any otherpolypeptide sequences that comprises different target recognitionsequences (e.g., including affibodies, peptibodies and other Fcfusions).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxyl terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region; this region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of sFv, see Pluckthun, The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Malmborg et al., J. Immunol. Methods 183:7-13,1995.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “one-armed antibody” or “one-armed antibodies” refers to anantibody that comprises (1) a variable domain joined by a peptide bondto a polypeptide comprising a CH2 domain, a CH3 domain or a CH2-CH3domain and (2) a second CH2, CH3 or CH2-CH3 domain, wherein a variabledomain is not joined by a peptide bond to a polypeptide comprising thesecond CH2, CH3 or CH2-CH3 domain. In one embodiment, the one-armedantibody comprises 3 polypeptides (1) a first polypeptide comprising avariable domain (e.g., VH), CH1, CH2 and CH3, (2) a second polypeptidecomprising a variable domain (e.g., VL) and a CL domain, and (3) a thirdpolypeptide comprising a CH2 and CH3 domain. In an embodiment, the thirdpolypeptide does not comprise a variable domain. In another embodiment,the one-armed antibody has a partial hinge region containing the twocysteine residues which form disulfide bonds linking the constant heavychains. In one embodiment, the variable domains of the one armedantibody form an antigen binding region. In another embodiment, avariable domain of the one armed antibody is a single variable domain,wherein each single variable domain is an antigen binding region.

Antibodies of the invention can be “chimeric” antibodies in which aportion of the heavy and/or light chain is identical with or homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, provided that they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interestherein include primatized antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey, Ape, etc.) and human constant region sequences.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Complex” or “complexed” as used here in refers to the association oftwo or more molecules that interact with each other through bonds and/orforces (e.g., van der Waals, hydrophobic, hydrophilic forces) that arenot peptide bonds. In one embodiment, the complex is heteromultimeric.It should be understood that the term “protein complex” or “polypeptidecomplex” as used herein includes complexes that have a non-proteinentity conjugated to a protein in the protein complex (e.g., including,but not limited to, chemical molecules such as a toxin or a detectionagent).

The term “heteromultimer” or “heteromultimeric” as used herein describestwo or more polypeptides that interact with each other by anon-peptidic, covalent bond (e.g., disulfide bond) and/or a non-covalentinteraction (e.g., hydrogen bonds, ionic bonds, Van der Waals forces,and hydrophobic interactions), wherein at least two of the moleculeshave different sequences from each other.

As used herein, “heteromultimerization domain” refers to alterations oradditions to a biological molecule so as to promote heteromultimerformation and hinder homomultimer formation. Any heterodimerizationdomain having a strong preference for forming heterodimers overhomodimers is within the scope of the invention. Illustrative examplesinclude but are not limited to, for example, US Patent Application20030078385 (Arathoon et al.—Genentech; describing knob into holes);WO2007147901 (Kjrgaard et al.—Novo Nordisk: describing ionicinteractions); WO 2009089004 (Kannan et al.—Amgen: describingelectrostatic steering effects); WO 2010/034605 (Christensen etal.—Genentech; describing coiled coils). See also, for example, Pack, P.& Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing leucinezipper or Pack et al., Bio/Technology 11, 1271-1277 (1993) describingthe helix-turn-helix motif. The phrase “heteromultimerization domain”and “heterodimerization domain” are used interchangeably herein. Incertain embodiments, the hinge-containing polypeptide comprises one ormore heterodimerization domains.

As used herein, the term “immunoadhesin” designates molecules whichcombine the binding specificity of a heterologous protein (an “adhesin”)with the effector functions of immunoglobulin constant domains.Structurally, the immunoadhesins comprise a fusion of an amino acidsequence with a desired binding specificity, which amino acid sequenceis other than the antigen recognition and binding site of an antibody(i.e., is “heterologous” compared to a constant region of an antibody),and an immunoglobulin constant domain sequence (e.g., CH2 and/or CH3sequence of an IgG). Exemplary adhesin sequences include contiguousamino acid sequences that comprise a portion of a receptor or a ligandthat binds to a protein of interest. Adhesin sequences can also besequences that bind a protein of interest, but are not receptor orligand sequences (e.g., adhesin sequences in peptibodies). Suchpolypeptide sequences can be selected or identified by various methods,include phage display techniques and high throughput sorting methods.The immunoglobulin constant domain sequence in the immunoadhesin can beobtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

An antibody of this invention “which binds” an antigen of interest isone that binds the antigen with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetinga protein or a cell or tissue expressing the antigen, and does notsignificantly cross-react with other proteins. In such embodiments, theextent of binding of the antibody to a “non-target” protein will be lessthan about 10% of the binding of the antibody to its particular targetprotein as determined by fluorescence activated cell sorting (FACS)analysis or radioimmunoprecipitation (RIA) or ELISA. With regard to thebinding of an antibody to a target molecule, the term “specific binding”or “specifically binds to” or is “specific for” a particular polypeptideor an epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction (e.g., anon-specific interaction may be binding to bovine serum albumin orcasein). Specific binding can be measured, for example, by determiningbinding of a molecule compared to binding of a control molecule. Forexample, specific binding can be determined by competition with acontrol molecule that is similar to the target, for example, an excessof non-labeled target. In this case, specific binding is indicated ifthe binding of the labeled target to a probe is competitively inhibitedby excess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about 200nM, alternatively at least about 150 nM, alternatively at least about100 nM, alternatively at least about 60 nM, alternatively at least about50 nM, alternatively at least about 40 nM, alternatively at least about30 nM, alternatively at least about 20 nM, alternatively at least about10 nM, alternatively at least about 8 nM, alternatively at least about 6nM, alternatively at least about 4 nM, alternatively at least about 2nM, alternatively at least about 1 nM, or greater. In one embodiment,the term “specific binding” refers to binding where a molecule binds toa particular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). For example, the Kd can be about 200 nM, 150nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM,2 nM, 1 nM, or stronger. Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by using surface plasmon resonance assays using a BIAcore™-2000or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J.Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J.Mol. Biol. 293:865-881 (1999). However, if the on-rate exceeds 10⁶ M⁻¹S⁻¹ by the surface plasmon resonance assay above, then the on-rate ispreferably determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophotometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

“Biologically active” and “biological activity” and “biologicalcharacteristics” with respect to a polypeptide of this invention, suchas an antibody, fragment, or derivative thereof, means having theability to bind to a biological molecule, except where specifiedotherwise.

“Peptibody” or “peptibodies” refers to a fusion of randomly generatedpeptides with an Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9,2003 to Feige et al. (incorporated by reference in its entirety). Theyinclude one or more peptides linked to the N-terminus, C-terminus, aminoacid sidechains, or to more than one of these sites. Peptibodytechnology enables design of therapeutic agents that incorporatepeptides that target one or more ligands or receptors, tumor-homingpeptides, membrane-transporting peptides, and the like. Peptibodytechnology has proven useful in design of a number of such molecules,including linear and disulfide-constrained peptides, “tandem peptidemultimers” (i.e., more than one peptide on a single chain of an Fcdomain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No.2003/0195156, published Oct. 16, 2003 (corresponding to WO 02/092620,published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, publishedSep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003);U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023,published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S.Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding toPCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filedSep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004),each of which is hereby incorporated by reference in its entirety.

“Affibodies” or “Affibody” refers to the use of a protein liked bypeptide bond to an Fc region, wherein the protein is used as a scaffoldto provide a binding surface for a target molecule. The protein is oftena naturally occurring protein such as staphylococcal protein A orIgG-binding B domain, or the Z protein derived therefrom (see Nilsson etal (1987), Prot Eng 1, 107-133, and U.S. Pat. No. 5,143,844) or afragment or derivative thereof. For example, affibodies can be createdfrom Z proteins variants having altered binding affinity to targetmolecule(s), wherein a segment of the Z protein has been mutated byrandom mutagenesis to create a library of variants capable of binding atarget molecule. Examples of affibodies include U.S. Pat. No. 6,534,628,Nord K et al, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech15:772-777 (1997). Biotechnol Appl Biochem. 2008 June; 50(Pt 2):97-112.

“Isolated” heteromultimer or complex means a heteromultimer or complexwhich has been separated and/or recovered from a component of itsnatural cell culture environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the heteromultimer, and may include enzymes,hormones, and other proteinaceous or nonproteinaceous solutes. Inpreferred embodiments, the heteromultimer will be purified (1) togreater than 95% by weight of protein as determined by the Lowry method,and most preferably more than 99% by weight, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain.

The heteromultimers of the present invention are generally purified tosubstantial homogeneity. The phrases “substantially homogeneous”,“substantially homogeneous form” and “substantial homogeneity” are usedto indicate that the product is substantially devoid of by-productsoriginated from undesired polypeptide combinations (e.g. homomultimers).

Expressed in terms of purity, substantial homogeneity means that theamount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2% or1% by weight or is less than 1% by weight. In one embodiment, theby-product is below 5%.

“Biological molecule” refers to a nucleic acid, a protein, acarbohydrate, a lipid, and combinations thereof. In one embodiment, thebiologic molecule exists in nature.

“Isolated,” when used to describe the various antibodies disclosedherein, means an antibody that has been identified and separated and/orrecovered from a cell or cell culture from which it was expressed.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and can include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes antibodies in situ withinrecombinant cells, because at least one component of the polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

By “linked” or “links” as used herein is meant either a direct peptidebond linkage between a first and second amino acid sequence or a linkagethat involves a third amino acid sequence that is peptide bonded to andbetween the first and second amino acid sequences. For example, a linkerpeptide bonded to the C-terminal end of one amino acid sequence and tothe N-terminal end of the other amino acid sequence.

By “linker” as used herein is meant an amino acid sequence of two ormore amino acids in length. The linker can consist of neutral polar ornonpolar amino acids. A linker can be, for example, 2 to 100 amino acidsin length, such as between 2 and 50 amino acids in length, for example,3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Alinker can be “cleavable,” for example, by auto-cleavage, or enzymaticor chemical cleavage. Cleavage sites in amino acid sequences and enzymesand chemicals that cleave at such sites are well known in the art andare also described herein.

By a “tether” as used herein is meant an amino acid linker that joinstwo other amino acid sequences. A tether as described herein can linkthe N-terminus of an immunoglobulin heavy chain variable domain with theC-terminus of an immunoglobulin light chain constant domain. Inparticular embodiments, a tether is between about 15 and 50 amino acidsin length, for example, between 20 and 26 amino acids in length (e.g.,20, 21, 22, 23, 24, 25, or 26 amino acids in length). A tether may be“cleavable,” for example, by auto-cleavage, or enzymatic or chemicalcleavage using methods and reagents standard in the art. In certainparticular embodiments, the tether comprises Gly-Gly-Ser repeats.

Enzymatic cleavage of a “linker” or a “tether” may involve the use of anendopeptidase such as, for example, Lys-C, Asp-N, Arg-C, V8, Glu-C,chymotrypsin, trypsin, pepsin, papain, thrombin, Genenase, Factor Xa,TEV (tobacco etch virus cysteine protease), Enterokinase, HRV C3 (humanrhinovirus C3 protease), Kininogenase, as well as subtilisin-likeproprotein convertases (e.g., Furin (PC1), PC2, or PC3) or N-argininedibasic convertase. Chemical cleavage may involve use of, for example,hydroxylamine, N-chlorosuccinimide, N-bromosuccinimide, or cyanogenbromide.

A “Lys-C endopeptidase cleavage site” as used herein is a Lysine residuein an amino acid sequence that can be cleaved at the C-terminal side byLys-C endopeptidase. Lys-C endopeptidase cleaves at the C-terminal sideof a Lysine residue. In certain embodiments, the half-antibody furthercomprises a K222A mutation in the hinge region to remove the endogenousLys-C endopeptidase cleavage site to preserve the structure of thehalf-antibody or the assembled bispecific antibody upon cleavage of thetether by Lys-C endopeptidase.

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S—S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. Prior to the present invention,FcγR binding was generally attributed to amino acid residues in thelower hinge region of an IgG Fc region.

The “CH2 domain” of a human IgG Fc region usually extends from aboutresidues 231 to about 340 of the IgG. The CH2 domain is unique in thatit is not closely paired with another domain. Rather, two N-linkedbranched carbohydrate chains are interposed between the two CH2 domainsof an intact native IgG molecule. It has been speculated that thecarbohydrate may provide a substitute for the domain-domain pairing andhelp stabilize the CH2 domain. Burton, Molec. Immunol. 22:161-206(1985).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG).

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc complex” as used herein refers to two CH2 domains of an Fc regioninteracting together and/or two CH3 domains of an Fc region interactingtogether, wherein the CH2 domains and/or the CH3 domains interactthrough bonds and/or forces (e.g., van der Waals, hydrophobic,hydrophilic forces) that are not peptide bonds.

“Fc component” as used herein refers to a hinge region, a CH2 domain ora CH3 domain of an Fc region.

“Fc CH component” or “FcCH” as used here in refers to a polypeptidecomprising a CH2 domain, a CH3 domain, or CH2 and CH3 domains of an Fcregion.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

In the present invention, “a reducing condition” is defined based on theredox potential in a reaction (for example in an assembly mixture) tomean that the redox potential of the reaction is negative (−). The redoxpotential of the reaction under reducing conditions is preferablybetween about −50 to −600 mV, −100 to −600 mV, −200 to −600 mV, −100 to−500 mV, −150 to −300 mV, more preferably between about −300 to −500 mV,most preferably about −400 mV.

Any suitable methods can be used to prepare a desired reducingcondition. For example, a desired reducing condition can be prepared byadding a reductant/reducing agent to the reaction (such as an assemblymixture of the invention). Suitable reductants include withoutlimitation dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP),thioglycolic acid, ascorbic acid, thiol acetic acid, glutathione (GSH),Beta-MercaptoEthylAmine, cysteine/cystine, GSH/glutathione disulfide(GSSG), cysteamine/cystamine, glycylcysteine, and beta-mercaptoethanol,preferably GSH. In certain particular embodiments, the reductant is aweak reductant including without limitation GSH,Beta-MercaptoEthylAmine, cysteine/cystine, GSH/GSSG,cysteamine/cystamine, glycylcysteine, and beta-mercaptoethanol,preferably GSH. In certain preferred embodiments, the reductant is GSH.It is within the ability of one of ordinary skill in the art to selectsuitable reductants at suitable concentrations and under suitableexperimental conditions to achieve in a reaction the desired reducingcondition. For example, 10 mM L-reduced glutathione in a solution with abispecific antibody protein concentration of 10 g/L at 20° C. willresult in a starting redox potential of about −400 mV. One of skill inthe art can use any suitable methods to measure the redox potential in agiven reaction.

The reducing condition of the reaction can be estimated and measuredusing any suitable methods known in the art. For example, the reducingcondition can be measured using a resazurin indicator (discolorizationfrom blue to colorless in reducing conditions). For more precisemeasurement, a redox-potential meter (such as an ORP Electrode made byBROADLEY JAMES®) can be used.

Alternatively, a reducing condition can be prepared by removingdissolved gases, especially dissolved oxygen, under reduced pressure ofabout 10 mmHg or less, preferably about 5 mmHg or less, more preferablyabout 3 mmHg or less, for about 1 to 60 minutes, preferably for about 5to 40 minutes.

In the present invention, it is preferred that reducing conditions aremaintained from immediately after mixing the first and secondhinge-containing polypeptides (such as half-antibodies) throughout theassembly step. In certain embodiments, the reaction or the assemblymixture is maintained in reducing conditions preferably for about 50% ormore, more preferably for about 70% or more, further more preferably forabout 90% or more of the reaction time. It is particularly preferredthat the redox-potential of the reaction medium is maintained from about−200 to −600 mV, more preferably between −300 to −500 mV, mostpreferably about −400 mV, for about 50% or more, more preferably forabout 70% or more, further more preferably for about 90% or more of thereaction time.

In certain particular embodiments, the reducing condition is a weakreducing condition. The term “weak reductant” or “weak reducingcondition” as used herein refers to a reducing agent or a reducingcondition prepared by the reducing agent having a negative oxidationpotential at 25° C. The oxidation potential of the reductant ispreferably between −50 to −600 mV, −100 to −600 mV, −200 to −600 mV,−100 to −500 mV, −150 to −300 mV, more preferably between about −300 to−500 mV, most preferably about −400 mV, when the pH is between 7 and 9,and the temperature is between 15° C. and 39° C. One skilled in the artwill be able to select suitable reductants for preparing a desiredreducing condition. The skilled researcher will recognize that a strongreductant, i.e., one that has a more negative oxidation potential thanabove mentioned reductants for the same concentration, pH andtemperature, may be used at a lower concentration. In a preferredembodiment, the proteins will be able to form disulfide bonds in thepresence of the reductant when incubated under the above-recitedconditions. Examples of a weak reductant include without limitationglutathione, Beta-MercaptoEthylAmine, cystine/cysteine, GSH/GSSG,cysteamine/cystamine, glycylcysteine, and beta-mercaptoethanol. Incertain embodiments, an oxidation potential similar to that of 200×molar ratio of GSH:Antibody can be used as a point of reference for aweakly reducing condition at which efficient assembly using otherreductants can be expected.

An “assembly mixture” is a solution comprising a first hinge-containingpolypeptide, a second hinge-containing polypeptide. In certainembodiments, the assembly mixture is present in a reducing condition. Insome embodiments, the assembly mixture is present in a weak reducingcondition. In certain other embodiments, the assembly mixture furthercomprises a weak reductant. The oxidation potential of the assemblymixture is between −50 to −600 mV, −100 to −600 mV, −200 to −600 mV,−100 to −500 mV, −150 to −300 mV, more preferably between about −300 to−500 mV, most preferably about −400 mV, when the pH is between 7 and 9,and the temperature is between 15° C. and 39° C.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,NY, 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc., NY, 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

II. Construction of Heteromultimeric Proteins

Typically, the heteromultimeric proteins described herein will comprisea significant portion of an antibody Fc region. In other aspects,however, the heavy chain comprises only a portion of the C_(H)1, C_(H)2,and/or C_(H)3 domains.

Heteromultimerization Domains

The heteromultimeric proteins comprise a heteromultimerization domain.To generate a substantially homogeneous population of heterodimericmolecule, the heterodimerization domain must have a strong preferencefor forming heterodimers over homodimers. Although the heteromultimericproteins exemplified herein use the knobs into holes technology tofacilitate heteromultimerization those skilled in the art willappreciate other heteromultimerization domains useful in the instantinvention.

Knobs into Holes

The use of knobs into holes as a method of producing multispecificantibodies is well known in the art. See U.S. Pat. No. 5,731,168 granted24 Mar. 1998 assigned to Genentech, PCT Pub. No. WO2009089004 published16 Jul. 2009 and assigned to Amgen, and US Pat. Pub. No. 20090182127published 16 Jul. 2009 and assigned to Novo Nordisk A/S. See also Marvinand Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann(2005) Acta Pharacol. Sin., 26:1-9. A brief discussion is provided here.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g. by alteringnucleic acid encoding the interface). Normally, nucleic acid encodingthe interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The upper limit for thenumber of original residues which are replaced is the total number ofresidues in the interface of the first polypeptide. The side chainvolumes of the various amino residues are shown in the following table.

TABLE 1 Properties of Amino Acid Residues Accessible One-Letter SurfaceAbbre- MASS^(a) VOLUME^(b) Area^(c) Amino Acid viation (daltons)(Angstrom³) (Angstrom²) Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R156.20 173.4 225 Asparagine (Asn) N 114.11 117.7 160 Aspartic acid (Asp)D 115.09 111.1 150 Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q128.14 143.9 180 Glutamic acid (Glu) E 129.12 138.4 190 Glycine (Gly) G57.06 60.1 75 Histidine (His) H 137.15 153.2 195 Isoleucine (Ile) I113.17 166.7 175 Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18168.6 200 Methionine (Met) M 131.21 162.9 185 Phenylalinine (Phe) F147.18 189.9 210 Proline (Pro) P 97.12 122.7 145 Serine (Ser) S 87.0889.0 115 Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21227.8 255 Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0155 ^(a)Molecular weight amino acid minus that of water. Values fromHandbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical RubberPublishing Co., 1961. ^(b)Values from A.A. Zamyatnin, Prog. Biophys.Mol. Biol. 24: 107-123, 1972. ^(c)Values from C. Chothia, J. Mol. Biol.105: 1-14, 1975. The accessible surface area is defined in FIGS. 6-20 ofthis reference.

The preferred import residues for the formation of a protuberance aregenerally naturally occurring amino acid residues and are preferablyselected from arginine (R), phenylalanine (F), tyrosine (Y) andtryptophan (W). Most preferred are tryptophan and tyrosine. In oneembodiment, the original residue for the formation of the protuberancehas a small side chain volume, such as alanine, asparagine, asparticacid, glycine, serine, threonine or valine. Exemplary amino acidsubstitutions in the CH3 domain for forming the protuberance includewithout limitation the T366W substitution.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). Normally, nucleic acid encoding the interface of the secondpolypeptide is altered to encode the cavity. To achieve this, thenucleic acid encoding at least one “original” amino acid residue in theinterface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue.The upper limit for the number of original residues which are replacedis the total number of residues in the interface of the secondpolypeptide. The side chain volumes of the various amino residues areshown in Table 1 above. The preferred import residues for the formationof a cavity are usually naturally occurring amino acid residues and arepreferably selected from alanine (A), serine (S), threonine (T) andvaline (V). Most preferred are serine, alanine or threonine. In oneembodiment, the original residue for the formation of the cavity has alarge side chain volume, such as tyrosine, arginine, phenylalanine ortryptophan. Exemplary amino acid substitutions in the CH3 domain forgenerating the cavity include without limitation the T366S, L368A,Y407A, Y407T and Y407V substitutions. In certain embodiments, the knobhalf-antibody comprises T366W substitution, and the hole half-antibodycomprises the T366S/L368A/Y407V substitutions.

An “original” amino acid residue is one which is replaced by an “import”residue which can have a smaller or larger side chain volume than theoriginal residue. The import amino acid residue can be a naturallyoccurring or non-naturally occurring amino acid residue, but preferablyis the former. “Naturally occurring” amino acid residues are thoseresidues encoded by the genetic code and listed in Table 1 above. By“non-naturally occurring” amino acid residue is meant a residue which isnot encoded by the genetic code, but which is able to covalently bindadjacent amino acid residue(s) in the polypeptide chain. Examples ofnon-naturally occurring amino acid residues are norleucine, ornithine,norvaline, homoserine and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym. 202:301-336 (1991), forexample. To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244: 182 (1989) and Ellman etal., supra can be used. Briefly, this involves chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA. Themethod of the instant invention involves replacing at least one originalamino acid residue, but more than one original residue can be replaced.Normally, no more than the total residues in the interface of the firstor second polypeptide will comprise original amino acid residues whichare replaced. Typically, original residues for replacement are “buried”.By “buried” is meant that the residue is essentially inaccessible tosolvent. Generally, the import residue is not cysteine to preventpossible oxidation or mispairing of disulfide bonds.

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity relies on modeling the protuberance/cavity pair based upon athree-dimensional structure such as that obtained by X-raycrystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

By “original or template nucleic acid” is meant the nucleic acidencoding a polypeptide of interest which can be “altered” (i.e.genetically engineered or mutated) to encode a protuberance or cavity.The original or starting nucleic acid may be a naturally occurringnucleic acid or may comprise a nucleic acid which has been subjected toprior alteration (e.g. a humanized antibody fragment). By “altering” thenucleic acid is meant that the original nucleic acid is mutated byinserting, deleting or replacing at least one codon encoding an aminoacid residue of interest. Normally, a codon encoding an original residueis replaced by a codon encoding an import residue. Techniques forgenetically modifying a DNA in this manner have been reviewed inMutagenesis: a Practical Approach, M. J. McPherson, Ed., (IRL Press,Oxford, UK. (1991), and include site-directed mutagenesis, cassettemutagenesis and polymerase chain reaction (PCR) mutagenesis, forexample. By mutating an original/template nucleic acid, anoriginal/template polypeptide encoded by the original/template nucleicacid is thus correspondingly altered.

The protuberance or cavity can be “introduced” into the interface of afirst or second polypeptide by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. Accordingly, the protuberance or cavity which is“introduced” is “non-naturally occurring” or “non-native”, which meansthat it does not exist in nature or in the original polypeptide (e.g. ahumanized monoclonal antibody).

Generally, the import amino acid residue for forming the protuberancehas a relatively small number of “rotamers” (e.g. about 3-6). A“rotamer” is an energetically favorable conformation of an amino acidside chain. The number of rotamers of the various amino acid residuesare reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

III. Vectors, Host Cells and Recombinant Methods

For recombinant production of a heteromultimeric protein (e.g., abispecific antibody) of the invention, the nucleic acid encoding it isisolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Manyvectors are available. The choice of vector depends in part on the hostcell to be used. Generally, preferred host cells are of eitherprokaryotic or eukaryotic (generally mammalian, but also including fungi(e.g., yeast), insect, plant, and nucleated cells from othermulticellular organisms) origin. It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species. In certain embodiments,the constant region is from IgG, particularly IgG1, IgG2 or IgG4.

A host cell is engineered such that it expresses a hinge-containingpolypeptide comprising a heterodimerization domain wherein the host celldoes not express a hinge-containing polypeptide comprising a secondheterodimerization domain.

a. Generating Heteromultimeric Proteins Using Prokaryotic Host Cells

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of theheteromultimeric proteins (e.g., an antibody) of the invention can beobtained using standard recombinant techniques. Desired polynucleotidesequences may be isolated and sequenced from, for example, antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used for the purpose of the presentinvention. Selection of an appropriate vector will depend mainly on thesize of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λEM.TM.-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. An inducible promoteris a promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding, for example, the light or heavy chain by removingthe promoter from the source DNA via restriction enzyme digestion andinserting the isolated promoter sequence into the vector of theinvention. Both the native promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of thetarget genes. In some embodiments, heterologous promoters are utilized,as they generally permit greater transcription and higher yields of theexpressed target gene as compared to the native target polypeptidepromoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker to operably ligate them to cistrons encoding the genes of theheteromultimeric protein, e.g., the target light and heavy chains(Siebenlist et al., (1980) Cell 20: 269), using linkers or adaptors tosupply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the signal sequences native to theheterologous polypeptides, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA andMBP. In one embodiment of the invention, the signal sequences used inboth cistrons of the expression system are STII signal sequences orvariants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. See Proba and Pluckthun Gene, 159:203(1995).

Prokaryotic host cells suitable for expressing heteromultimeric proteins(e.g., antibodies) of the invention include Archaebacteria andEubacteria, such as Gram-negative or Gram-positive organisms. Examplesof useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g.,B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa),Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus,Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment,gram-negative cells are used. In one embodiment, E. coli cells are usedas hosts for the invention. Examples of E. coli strains include strainW3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington,D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCCDeposit No. 27,325) and derivatives thereof, including strain 33D3having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE)degP41 kan^(R) (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ) 1776(ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. In oneembodiment, E. coli A/pp finds particular use. These examples areillustrative rather than limiting. Methods for constructing derivativesof any of the above-mentioned bacteria having defined genotypes areknown in the art and described in, for example, Bass et al., Proteins,8:309-314 (1990). It is generally necessary to select the appropriatebacteria taking into consideration replicability of the replicon in thecells of a bacterium. For example, E. coli, Serratia, or Salmonellaspecies can be suitably used as the host when well-known plasmids suchas pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.Typically the host cell should secrete minimal amounts of proteolyticenzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Polypeptide Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniquecommonly used is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the first and second hinge-containing host cells arecultured separately and the expressed polypeptides of the presentinvention are secreted into and recovered from the periplasm of the hostcells separately. In a second embodiment, the first and secondhinge-containing host cells are cultured separately and prior to theisolation of the hinge-containing polypeptides, the two host cellcultures are mixed together and the cells pelleted. In a thirdembodiment, the first and second hinge-containing host cells arecultured separately, centrifuged and resuspended separately and thenmixed together prior to isolation of the hinge-containing polypeptides.In fourth embodiment, the first and second hinge-containing host cellsare cultured together in the same culture vessel. Protein recoverytypically involves disrupting the microorganism cell membrane, generallyby such means as osmotic shock, sonication or lysis. Once cells aredisrupted, cell debris or whole cells may be removed by centrifugationor filtration. The proteins may be further purified, for example, byaffinity resin chromatography. Alternatively, proteins can betransported or secreted into the culture media and isolated therein.Recombinant proteins expressed with an exogenous sequence tag (orepitope tag) can facilitate the purification step. The technique ofcloning and purification of proteins containing an exogenous sequencetag (including without limitation the His tag and GST tag) is well knownin the art. Cells may be removed from the culture and the culturesupernatant being filtered and concentrated for further purification ofthe proteins produced. The expressed polypeptides can be furtherisolated and identified using commonly known methods such aspolyacrylamide gel electrophoresis (PAGE) and Western blot assay. Theisolated polypeptides will be used to produce the heteromultimericproteins.

In one aspect of the invention, heteromultimeric protein (e.g.,antibody) production is conducted in large quantity by a fermentationprocess. Various large-scale fed-batch fermentation procedures areavailable for production of recombinant proteins. Large-scalefermentations have at least 1000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors use agitatorimpellers to distribute oxygen and nutrients, especially glucose (thepreferred carbon/energy source). Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secretedheteromultimeric proteins (e.g., antibodies), additional vectorsoverexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB,DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerasewith chaperone activity) can be used to co-transform the hostprokaryotic cells. The chaperone proteins have been demonstrated tofacilitate the proper folding and solubility of heterologous proteinsproduced in bacterial host cells. Chen et al. (1999) J Bio Chem274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou etal., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol.Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),Proc. Natl. Acad. Sci. USA 95:2773-2777; Georgiou et al., U.S. Pat. No.5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al.,Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention. In asecond embodiment, the E. coli strain is deficient for a lipoprotein ofthe outer membrane (Alpp).

iii. Heteromultimeric Protein Purification

In one embodiment, the heteromultimeric protein produced herein isfurther purified to obtain preparations that are substantiallyhomogeneous for further assays and uses. Standard protein purificationmethods known in the art can be employed. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC, chromatography on silica or on an ion-exchange resin such asDEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, andgel filtration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of, for example, the half-antibody or fulllength antibody products of the invention. Protein A is a 41 kD cellwall protein from Staphylococcus aureus which binds with a high affinityto the Fc region of antibodies. Lindmark et al. (1983) J. Immunol. Meth.62:1-13. The solid phase to which Protein A is immobilized is preferablya column comprising a glass or silica surface, more preferably acontrolled pore glass column or a silicic acid column. In someapplications, the column has been coated with a reagent, such asglycerol, in an attempt to prevent nonspecific adherence ofcontaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. The heteromultimeric protein(e.g., antibody) is recovered from the solid phase by elution.

b. Generating Heteromultimeric Proteins Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

i. Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available. The DNA for such precursor region is ligated inreading frame to DNA encoding the desired heteromultimeric protein(s)(e.g., antibodies).

ii. Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used, but only because it contains the early promoter.

iii. Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, for example, U.S. Pat. No. 4,965,199.

iv. Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the desiredhinge-containing polypeptide(s) (e.g., antibody) nucleic acid. Promotersequences are known for eukaryotes. Virtually all eukaryotic genes havean AT-rich region located approximately 25 to 30 bases upstream from thesite where transcription is initiated. Another sequence found 70 to 80bases upstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Desired hinge-containing polypeptide(s) (e.g., half-antibody)transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such as, forexample, polyoma virus, fowlpox virus, adenovirus (such as Adenovirus2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, or from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

v. Enhancer Element Component

Transcription of DNA encoding the desired hinge-containingpolypeptide(s) (e.g., antibody) by higher eukaryotes can be increased byinserting an enhancer sequence into the vector. Many enhancer sequencesare now known from mammalian genes (e.g., globin, elastase, albumin,α-fetoprotein, and insulin genes). Also, one may use an enhancer from aeukaryotic cell virus. Examples include the SV40 enhancer on the lateside of the replication origin (bp 100-270), the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. See also Yaniv, Nature297:17-18 (1982) for a description of elements for enhancing activationof eukaryotic promoters. The enhancer may be spliced into the vector ata position 5′ or 3′ to the antibody polypeptide-encoding sequence,provided that enhancement is achieved, but is generally located at asite 5′ from the promoter.

vi. Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

vii. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for desired hinge-containing polypeptide(s) (e.g.,antibody) production and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

viii. Culturing the host cells

The host cells used to produce a desired hinge-containing polypeptide(s)(e.g., antibody) of this invention may be cultured in a variety ofmedia. Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

ix. Purification of Heteromultimeric Proteins

When using recombinant techniques, the hinge-containing polypeptides canbe produced intracellularly, or directly secreted into the medium. Ifthe hinge-containing polypeptide is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Where thehinge-containing polypeptide is secreted into the medium, supernatantsfrom such expression systems are generally first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. A protease inhibitorsuch as PMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The heteromultimer composition prepared from the cells can be purifiedusing, for example, ion exchange, hydrophobic interactionchromatography, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. Combinations of theabove-mentioned techniques are also contemplated. The suitability ofprotein A as an affinity ligand depends on the species and isotype ofany immunoglobulin Fc domain that is present in the antibody. Protein Acan be used to purify antibodies that are based on human γ1, γ2, or γ4heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).Protein G is recommended for all mouse isotypes and for human γ3 (Gusset al., EMBO J. 5:15671575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt). The production of the heteromultimericproteins can alternatively or additionally (to any of the foregoingparticular methods) comprise dialyzing a solution comprising a mixtureof the polypeptides.

x. Antibody Production Using Baculovirus

Recombinant baculovirus may be generated by co-transfecting a plasmidencoding an antibody or antibody fragment and BaculoGold™ virus DNA(Pharmingen) into an insect cell such as a Spodoptera frugiperda cell(e.g., Sf9 cells; ATCC CRL 1711) or a Drosophila melanogaster S2 cellusing, for example, lipofectin (commercially available from GIBCO-BRL).In a particular example, an antibody sequence is fused upstream of anepitope tag contained within a baculovirus expression vector. Suchepitope tags include poly-His tags. A variety of plasmids may beemployed, including plasmids derived from commercially availableplasmids such as pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly,the sequence encoding an antibody or a fragment thereof may be amplifiedby PCR with primers complementary to the 5′ and 3′ regions. The 5′primer may incorporate flanking (selected) restriction enzyme sites. Theproduct may then be digested with the selected restriction enzymes andsubcloned into the expression vector.

After transfection with the expression vector, the host cells (e.g., Sf9cells) are incubated for 4-5 days at 28° C. and the released virus isharvested and used for further amplifications. Viral infection andprotein expression may be performed as described, for example, byO'Reilley et al. (Baculovirus expression vectors: A Laboratory Manual.Oxford: Oxford University Press (1994)).

Expressed poly-His tagged antibody can then be purified, for example, byNi2+-chelate affinity chromatography as follows. Extracts can beprepared from recombinant virus-infected Sf9 cells as described byRupert et al. (Nature 362:175-179 (1993)). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL HEPES pH 7.9; 12.5 mMMgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate; 300 mM NaCl; 10% glycerol pH 7.8) and filtered througha 0.45 μm filter. A Ni2+-NTA agarose column (commercially available fromQiagen) is prepared with a bed volume of 5 mL, washed with 25 mL ofwater, and equilibrated with 25 mL of loading buffer. The filtered cellextract is loaded onto the column at 0.5 mL per minute. The column iswashed to baseline A280 with loading buffer, at which point fractioncollection is started. Next, the column is washed with a secondary washbuffer (50 mM phosphate; 300 mM NaCl; 10% glycerol pH 6.0), which elutesnonspecifically bound protein. After reaching A280 baseline again, thecolumn is developed with a 0 to 500 mM Imidazole gradient in thesecondary wash buffer. One mL fractions are collected and analyzed bySDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated toalkaline phosphatase (Qiagen). Fractions containing the elutedHis10-tagged antibody are pooled and dialyzed against loading buffer.

Alternatively, purification of the antibody can be performed using knownchromatography techniques, including for instance, Protein A or proteinG column chromatography. In one embodiment, the antibody of interest maybe recovered from the solid phase of the column by elution into asolution containing a chaotropic agent or mild detergent. Exemplarychaotropic agents and mild detergents include, but are not limited to,Guanidine-HCl, urea, lithium perclorate, Arginine, Histidine, SDS(sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which arecommercially available.

IV. Target Molecules

Examples of molecules that may be targeted by a heteromultimeric proteinof this invention include, but are not limited to, soluble serumproteins and their receptors and other membrane bound proteins (e.g.,adhesins). See WO2011/133886, which is incorporated by reference hereinin its entirety.

In another embodiment the heteromultimeric protein of the invention iscapable of binding one, two or more cytokines, cytokine-relatedproteins, and cytokine receptors selected from the group consisting ofBMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2(GM-CSF), CSF3 (G-CSF), EPO, FGFI (aFGF), FGF2 (bFGF), FGF3 (int-2),FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF1 1, FGF12,FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2,IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNBI, IFNG, IFNWI, FELI, FELI(EPSELON), FELI (ZETA), IL1A, IL1 B, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B,IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B,IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB,TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL),TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1 BB ligand),TNFSFIO (TRAIL), TNFSF1 I (TRANCE), TNFSF12 (APO3L), TNFSF13 (April),TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF,VEGFB, VEGFC, ILIR1, IL1 R2, IL1 RL1, IL1 RL2, IL2RA, IL2RB, IL2RG,IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILI0RA, ILI0RB,IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1,IL20RA, IL21 R, IL22R, IL1 HY1, IL1 RAP, IL1 RAPL1, IL1 RAPL2, IL1 RN,IL6ST, IL18BP, IL18RAP, IL22RA2, AIFI, HGF, LEP (leptin), PTN, and THPO.

In another embodiment, a target molecule is a chemokine, chemokinereceptor, or a chemokine-related protein selected from the groupconsisting of CCLI (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-Ia), CCL4(MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLH (eotaxin),CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21 (SLC/exodus-2), CCL22(MDC/STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCLI (GROI), CXCL2 (GRO2),CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP10), CXCL11 (I-TAC), CXCL12 (SDFI), CXCL13, CXCL14, CXCL16, PF4 (CXCL4),PPBP (CXCL7), CX3CL1 (SCYDI), SCYEI, XCLI (lymphotactin), XCL2 (SCM-Ib),BLRI (MDR15), CCBP2 (D6/JAB61), CCR1 (CKRI/HM145), CCR2 (mcp-IRB/RA),CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBII), CCR8(CMKBR8/TERI/CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR), XCRI(GPR5/CCXCRI), CMKLRI, CMKORI (RDCI), CX3CR1 (V28), CXCR4, GPR2 (CCRIO),GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo),HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCPIO, CKLFSF2,CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3,GRCCIO (CIO), EPO, FY (DARC), GDF5, HDFIA, DL8, PRL, RGS3, RGS13, SDF2,SLIT2, TLR2, TLR4, TREMI, TREM2, and VHL.

In another embodiment the heteromultime c proteins of the invention arecapable of binding one or more targets selected from the groupconsisting of ABCFI; ACVRI; ACVRIB; ACVR2; ACVR2B; ACVRLI; AD0RA2A;Aggrecan; AGR2; AICDA; AIFI; AIGI; AKAPI; AKAP2; AMH; AMHR2; ANGPTI;ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOCI; AR; AZGPI(zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAGI; BAH; BCL2;BCL6; BDNF; BLNK; BLRI (MDR15); BMPI; BMP2; BMP3B (GDFIO); BMP4; BMP6;BMP8; BMPRIA; BMPRIB; BMPR2; BPAGI (plectin); BRCAI; C19orfIO (IL27w);C3; C4A; C5; C5R1; CANTI; CASP1; CASP4; CAVI; CCBP2 (D6/JAB61); CCLI(1-309); CCLII (eotaxin); CCL13 (MCP-4); CCL15 (MIP-Id); CCL16 (HCC-4);CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20(MIP-3a); CCL21 (MTP-2); SLC; exodus-2; CCL22 (MDC/STC-I); CCL23(MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3);CCL27 (CTACK/ILC); CCL28; CCL3 (MTP-Ia); CCL4 (MDP-Ib); CCL5 (RANTES);CCL7 (MCP-3); CCL8 (mcp-2); CCNAI; CCNA2; CCNDI; CCNEI; CCNE2; CCR1(CKRI/HM145); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5(CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBII);CCR8 (CMKBR8/TERI/CKR-LI); CCR9 (GPR-9-6); CCRLI (VSHKI); CCRL2 (L-CCR);CD164; CD19; CDIC; CD20; CD200; CD22; CD24; CD28; CD3; CD37; CD38; CD3E;CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74;CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDHI (E-cadherin); CDH10;CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3;CDK4; CDK5; CDK6; CDK7; CDK9; CDKNIA (p21WapI/CipI); CDKNIB (p27KipI);CDKNIC; CDKN2A (P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERI; CHGA;CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin);CMKLRI; CMKORI (RDCI); CNRI; COL18A1; COLIAI; COL4A3; COL6A1; CR2; CRP;CSFI (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI (b-catenin);CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28); CXCLI (GROI); CXCL10(IP-10); CXCLII (I-TAC/IP-9); CXCL12 (SDFI); CXCL13; CXCL14; CXCL16;CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9(MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5;CYCI; CYSLTRI; DAB2IP; DES; DKFZp451 J01 18; DNCLI; DPP4; E2F1; ECGFI;EDGI; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3;EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESRI; ESR2; F3 (TF); FADD; FasL;FASN; FCERIA; FCER2; FCGR3A; FGF; FGFI (aFGF); FGF10; FGF11; FGF12;FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20;FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FELI (EPSILON); FILI (ZETA);FLJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI (FRA-I); FY(DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST; GATA3; GDF5; GFI1;GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80);GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPI; HAVCR2; HDAC4; HDAC5; HDAC7A;HDAC9; HGF; HIFIA; HDPI; histamine and histamine receptors; HLA-A;HLA-DRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNAI;IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; DFNWI; IGBPI; IGFI;IGFIR; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; MO; IL10RA; IL10RB; IL1 1;IL1 1 RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2;IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP;IL18R1; IL18RAP; IL19; IL1A; IL1 B; ILIF10; IL1 F5; IL1 F6; IL1 F7; IL1F8; IL1 F9; IL1 HYI; IL1 RI; IL1 R2; IL1 RAP; IL1 RAPL1; IL1 RAPL2; IL1RL1; IL1 RL2, ILIRN; IL2; IL20; IL20RA; IL21 R; IL22; IL22R; IL22RA2;IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG;IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA;INSL3; INSL4; IRAKI; ERAK2; ITGAI; ITGA2; ITGA3; ITGA6 (a6 integrin);ITGAV; ITGB3; ITGB4 (b 4 integrin); JAGI; JAKI; JAK3; JUN; K6HF; KAII;KDR; KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15;KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6(hair-specific type H keratin); LAMAS; LEP (leptin); Lingo-p75;Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR;MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIBI; midkine; MEF; MIP-2;MK167; (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III);MTSSI; MUCI (mucin); MYC; MYD88; NCK2; neurocan; NFKBI; NFKB2; NGFB(NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NMEI(NM23A); N0X5; NPPB; NROBI; NR0B2; NRIDI; NR1 D2; NR1 H2; NR1 H3; NR1H4; NR1 I2; NR1 I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6;NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRPI; NRP2;NT5E; NTN4; ODZI; OPRDI; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA;PDGFA; PDGFB; PECAMI; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG;PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7); PPID; PRI; PRKCQ; PRKDI; PRL;PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGSI; RGS13; RGS3; RNFIIO (ZNF144); ROBO2; S100A2; SCGB1 D2(lipophilin B); SCGB2A1 (mammaglobin2); SCGB2A2 (mammaglobin 1); SCYEI(endothelial Monocyte-activating cytokine); SDF2; SERPINAI; SERPINA3;SERP1 NB5 (maspin); SERPINEI (PAI-I); SERPDMF1; SHBG; SLA2; SLC2A2;SLC33A1; SLC43A1; SLIT2; SPPI; SPRRIB (SprI); ST6GAL1; STABI; STATE;STEAP; STEAP2; TB4R2; TBX21; TCPIO; TDGFI; TEK; TGFA; TGFBI; TGFBIII;TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; THIL; THBSI(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissuefactor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSFIA; TNFRSFIB; TNFRSF21;TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL);TNFSFI 1 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14(HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand);TNFSF9 (4-1 BB ligand); TOLLIP; Toll-like receptors; TOP2A(topoisomerase Ea); TP53; TPMI; TPM2; TRADD; TRAFI; TRAF2; TRAF3; TRAF4;TRAF5; TRAF6; TREMI; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC;versican; VHL C5; VLA-4; XCLI (lymphotactin); XCL2 (SCM-Ib);XCRI(GPR5/CCXCRI); YYI; and ZFPM2.

Preferred molecular target molecules for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD16, CD19,CD20, CD34; CD64, CD200 members of the ErbB receptor family such as theEGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules suchas LFA-1, Mad, p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, andalphav/beta3 integrin including either alpha or beta subunits thereof(e.g., anti-CD11a, anti-CD18 or anti-CD1 1b antibodies); growth factorssuch as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (alphalFN);TNFalpha, an interleukin, such as IL-1 beta, IL-3, IL-4, IL-5, IL-8,IL-9, IL-13, IL17A/F, IL-18, IL-13Ralpha1, IL13Ralpha2, IL-4R, IL-5R,IL-9R, IgE; blood group antigens; flk2/flt3 receptor; obesity (OB)receptor; mpI receptor; CTLA-4; RANKL, RANK, RSV F protein, protein Cetc.

In one embodiment, the heteromultimeric proteins of this invention bindlow-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 ortransferrin receptor, and at least one target selected from the groupconsisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3)gamma-secretase, 4) tau-secretase, 5) amyloid precursor protein (APP),6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) alpha-synuclein,9) Parkin, 10) Huntingtin, 1 1) p75 NTR, and 12) caspase-6.

In one embodiment, the heteromultimeric proteins of this invention bindsto at least two target molecules selected from the group consisting of:IL-1 alpha and IL-1 beta, IL-12 and IL-18; IL-13 and IL-9; IL-13 andIL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1 beta; IL-13 andIL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β;IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 andSPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS, IL-13 and PED2, IL17A andIL17F, CD3 and CD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20and CD3; CD38 and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20;CD-8 and IL-6; CD20 and BR3, TNFalpha and TGF-beta, TNFalpha and IL-1beta; TNFalpha and IL-2, TNF alpha and IL-3, TNFalpha and IL-4, TNFalphaand IL-5, TNFalpha and IL6, TNFalpha and IL8, TNFalpha and IL-9,TNFalpha and IL-10, TNFalpha and IL-1 1, TNFalpha and IL-12, TNFalphaand IL-13, TNFalpha and IL-14, TNFalpha and IL-15, TNFalpha and IL-16,TNFalpha and IL-17, TNFalpha and IL-18, TNFalpha and IL-19, TNFalpha andIL-20, TNFalpha and IL-23, TNFalpha and IFNalpha, TNFalpha and CD4,TNFalpha and VEGF, TNFalpha and MIF, TNFalpha and ICAM-1, TNFalpha andPGE4, TNFalpha and PEG2, TNFalpha and RANK ligand, TNFalpha and Te38;TNFalpha and BAFF; TNFalpha and CD22; TNFalpha and CTLA-4; TNFalpha andGP130; TNFa and IL-12p40; VEGF and HER2, VEGF-A and HER2, VEGF-A andPDGF, HER1 and HER2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5,VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR,HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR(HER1)and HER2, EGFR and HER3, EGFR and HER4, IL-13 and CD40L, IL4 and CD40L,TNFR1 and IL-1 R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3,MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTNO2; IGF1and IGF2; IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGMA; OMGp and RGM A; PDL-I and CTLA-4; and RGM A and RGM B.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g.,the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.,cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

V. Embodiments

The invention provides additional embodiments as described below. In afirst embodiment, a method of producing a heteromultimeric protein isprovided, said method comprising: Obtaining a protein A purified firsthinge-containing polypeptide; Obtaining a protein A purified secondhinge-containing polypeptide; Adjusting the pH of each half-antibody tobetween 4 and 9; Mixing the first and second hinge-containingpolypeptide to obtain a mixed hinge-containing polypeptide pool; Addinga molar excess of a weak reductant to the mixed hinge-containingpolypeptide pool; and incubating the mixed hinge-containing polypeptidepool with the weak reducant to form a heteromultimeric proteincomprising the first and second hinge-containing polypeptide.

In a second embodiment and according to the first embodiment, the firstand second hinge-containing polypeptides can be selected from ahalf-antibody, immunoadhesin and fragments thereof. In a thirdembodiment and according to the first embodiment, the firsthinge-containing polypeptide is a half-antibody. In a fourth embodimentand according to the first embodiment, the second hinge-containingpolypeptide is an Fc component. In a fifth embodiment and according tothe third embodiment, the half-antibody comprises a VL domain, a CLdomain, a VH domain, a CH1 domain, a hinge domain, a CH2 domain and aCH3 domain. In a sixth embodiment and according to the fifth embodiment,the half-antibody is a single polypeptide chain further comprises atether wherein said domains are positioned relative to each other in anN-terminal to C-terminal direction as follows:VL-CL-tether-VH-CH1-hinge-CH2-CH3. In a seventh embodiment and accordingto the first embodiment, the first and second hinge-containingpolypeptides are mixed prior to Protein A purification and co-purifiedover Protein A. In an eighth embodiment and according to the firstembodiment, the first and second hinge-containing polypeptides comprisea heteromultimerization domain. In a ninth embodiment and according tothe eighth embodiment, the heteromultimerization domain is selected froma knob into hole mutation, leucine zippers, electrostatic, etc. In atenth embodiment and according to the ninth embodiment, the firsthinge-containing polypeptide comprises a knob and the secondhinge-containing polypeptide comprises a hole. In an eleventh embodimentand according to the first embodiment, the pH is adjusted after mixing.In a twelfth embodiment and according to the first or eleventhembodiment, the method further comprises adding L-Arginine to a finalconcentration of between 20 mM to 1M prior to adjusting the pH. In athirteenth embodiment and according to the first embodiment, the methodfurther comprises incubating the mixed pool at a temperature of between15° C. and 39° C. for at least 30 minutes. In a fourteenth embodimentand according to the first embodiment, the assembly mixture has anoxidation potential of between −200 to −600 mV, more preferably between−300 to −500 mV, most preferably about −400 mV. In a fifteenthembodiment and according to the first embodiment, the weak reductant isselected from GSH, Beta-MercaptoEthylAmine, cysteine/cysteine, GSH/GSSG,cysteamine/cystamine, glycylcysteine, and beta-mercaptoethanol. In asixteenth embodiment and according to the first embodiment, the weakreductant is added in 50-600 molar excess. In a seventeenth embodimentand according to the first embodiment, the weak reductant is added priorto mixing. In an eighteenth embodiment and according to the seventeenthembodiment, the addition is done less than one hour prior to mixing. Ina nineteenth embodiment and according to the first embodiment, the stepof incubating the assembly mixture is done at a temperature between 15°C. and 39° C. in the presence of Polyvinylpyrrolidone (PVP). In atwentieth embodiment and according to the nineteenth embodiment,histidine is added prior to, simultaneously with or after the PVP. In a21^(st) embodiment and according to the nineteenth embodiment, the PVPis added up to 40% (w/v).

In a 22^(nd) embodiment, the invention provides a method of producing abispecific antibody, said method comprising:

-   -   a. Obtaining a protein A purified first half-antibody;    -   b. Obtaining a protein A purified second half-antibody;    -   c. Adding a L-Arginine solution to each half-antibody;    -   d. Adjusting the pH of each half-antibody to between 4 and 9;    -   e. Mixing the first and second half-antibody pools to obtain a        mixed half-antibody pool,    -   f. adding a molar excess of a weak reductant to the mixed        half-antibody pool;    -   g. incubating the mixed half-antibody pool at a temperature        between 15° C. and 39° C. in the presence of PVP,

whereby a bispecific antibody comprising the first and secondhalf-antibody is produced.

In a 23^(rd) embodiment, the invention provides a method of producing aheteromultimer, said method comprising: (a) Providing an L-argininecontaining mixture of at least two different hinge-containingpolypeptides, wherein said mixture has a pH of between 7 and 9, (b)adding a molar excess of a weak reductant and (c) incubating the mixtureunder conditions whereby a heteromultimer is produced.

In a 24^(th) embodiment and according to the 22^(nd) embodiment, thefirst half-antibody is a single chain polypeptide comprising (a) Afull-length Light chain comprising a VL domain and a CL domain; (b) Atether, (c) A full length Heavy chain comprising a VH domain, CH1domain, a hinge, a CH2 domain and a CH3 domain; said polypeptidecomprising domains of the light and heavy chains positioned relative toeach other in an N-terminal to C-terminal direction as follows:VL-CL-CL/VH tether-VH-CH1-hinge-CH2-CH3. In a 25^(th) embodiment andaccording to the 24^(nd) embodiment, the single chain polypeptidefurther comprises a heteromultimerization domain. In a 26^(th)embodiment and according to the 25^(th) embodiment, theheteromultimerization domain is either a hole (e.g., cavity) or knob(e.g., protuberance). In a 27^(th) embodiment and according to the26^(th) embodiment, the second half-antibody comprises a hole when thefirst half-antibody comprises a knob. In a 28^(th) embodiment andaccording to the 26^(th) embodiment, the second half-antibody comprisesa knob when the first half-antibody comprises a hole. In a 29^(th)embodiment and according to the 24^(th) embodiment, the tether comprisesGGS repeats. In a 30^(th) embodiment and according to the 24^(th)embodiment, the tether is 15-50 amino acids.

In a 31^(st) embodiment, the invention provides a method of producing aheteromultimeric protein, said method comprising: (a) Obtaining aprotein A purified first hinge-containing polypeptide; (b) Obtaining aprotein A purified second hinge-containing polypeptide; (c) Adjustingthe pH of each hinge-containing polypeptide to between 4 and 9 in thepresence of L-Arginine; (d) Mixing the first and second hinge-containingpolypeptide to obtain a mixed half-antibody pool, and incubating to forma heteromultimeric protein comprising the first and secondhinge-containing polypeptide.

In a 32^(nd) embodiment and according to the first or 31^(st)embodiment, at least one of the half-antibodies is a single chainpolypeptide comprising: (a) A full-length Light chain comprising a VLdomain and a CL domain; (b) A tether; (c) A full length Heavy chaincomprising a VH domain, CH1 domain, a hinge, a CH2 domain and a CH3domain.

In a 33^(rd) embodiment and according to the first embodiment, the firsthinge-containing polypeptide is a single chain polypeptide comprisingdomains of the light and heavy chains positioned relative to each otherin an N-terminal to C-terminal direction as follows:VL-CL-VH-CH1-hinge-CH2-CH3. In a 34^(th) embodiment and according to the33^(rd) embodiment, the single polypeptide chain further comprises atether wherein said domains are positioned relative to each other in anN-terminal to C-terminal direction as follows:VL-CL-tether-VH-CH1-hinge-CH2-CH3.

In a 35^(th) embodiment, the invention provides a method of producing aheteromultimer, said method comprising providing an L-argininecontaining a mixture of hinge-containing polypeptides, said mixturehaving a pH of between 4 and 9, adding a weak reductant and incubatingunder conditions so as to produce a heteromultimer.

In a 36^(th) embodiment, the invention provides a host cell that hasbeen engineered to express a half-antibody wherein said half-antibody isa single chain polypeptide comprising a tether, a VL domain, a CLdomain, a VH domain, a CH1 domain, a hinge domain, a CH2 domain and aCH3 domain wherein said domains are positioned relative to each other inan N-terminal to C-terminal direction as follows:VL-CL-tether-VH-CH1-hinge-CH2-CH3. In a 37^(th) embodiment and accordingto the 36^(th) embodiment, the single polypeptide chain furthercomprises a heterodimerization domain. In a 38^(th) embodiment andaccording to the 36^(th) or the 37^(th) embodiment, the host cell isselected from prokaryotic cells, eukaryotic cells, mammalian cells orplant cells. In a 39^(th) embodiment and according to the 38^(th)embodiment, the host cell is a prokaryotic cell. In a 40^(th) embodimentand according to the 39^(th) embodiment, the prokaryotic cell is an E.coli cell. In a 41^(st) embodiment and according to the 40^(th)embodiment, the E. coli cell is Ipp deficient. In a 42^(nd) embodimentand according to the 38^(th) embodiment, the host cell is a mammaliancell. In a 43^(rd) embodiment and according to the 41^(st) embodiment,the mammalian cell is a CHO cell. In a 44^(th) embodiment and accordingto the 36^(th) or 37^(th) embodiment, the host cell comprises a vectorencoding the single chain half-antibody. In a 45^(th) embodiment andaccording to the 36^(th) or 37^(th) embodiment, the half-antibodyfurther comprises a heterodimerization domain. In a 46^(th) embodimentand according to the 45^(th) embodiment, the heterodimerization domainis selected from a knob, a hole, one or more charged amino acids withinthe interface that are electrostatically unfavorable to homodimerformation but electrostatically favorable to heterodimer formation, oneor more amino acids are altered to enhance intramolecular ionicinteractions, a coiled coil and a leucine zipper.

In a 47^(th) embodiment, the invention provides a mixture of host cellscomprising a first host cell engineered to express a first single chainhalf-antibody and a second host cell engineered to express an Fccomponent. In a 48^(th) embodiment and according to the 36^(th)embodiment, the half-antibody produced by a host cell comprises aheterodimerization domain.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); pmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds).

EXAMPLES

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein.

Example 1 Expression & Purification

This example illustrates the expression and purification ofhalf-antibodies.

Exemplary methods of construction and expression of half-antibodies inE. coli can be found for example in co-pending application U.S.2011/0287009, which is incorporated herein by reference in its entirety.It is within the ability of one of skill in the art to modify and adjustthe culture and expression conditions.

Expression of Half-Antibodies in E. coli Cells

Construction of Expression Plasmids

Both the heavy and light chain DNA coding sequences were cloned into anexpression plasmid that contained separate promoter elements for each ofthe sequences and antibiotic resistance for selection of bacterial cellsthat contain the expression plasmid. The vector constructs also encodethe heat-stable enterotoxin II (STII) secretion signal (Picken et al.,1983, Infect. Immun. 42:269-275, and Lee et al., 1983, Infect. Immun.42:264-268) for the export of the antibody polypeptides into theperiplasmic space of the bacterial cell. Transcription of each chain iscontrolled by the phoA promoter (Kikuchi et al., 1981, Nucleic AcidsRes., 9:5671-5678) and translational control is provided by previouslydescribed STII signal sequence variants of measured relativetranslational strength, which contain silent codon changes in thetranslation initiation region (TIR) (Simmons and Yansura, 1996, NatureBiotechnol. 14:629-634 and Simmons et al., 2002, J. Immunol. Methods,263:133-147).

Each half-antibody had either a knob (protuberance) or a hole (cavity)engineered into the heavy chain as described in U.S. Pat. No. 7,642,228.Briefly, a CH3 knob mutant was generated first. A library of CH3 holemutants was then created by randomizing residues 366, 368 and 407 thatare in proximity to the knob on the partner CH3 domain. In the followingexamples, the knob mutation was T366W, and the hole had mutations T366S,L368A and Y407V in an IgG1 or IgG4 backbone. Equivalent mutations inother immunoglobulin isotypes can be made by one skilled in the art.Further, the skilled artisan will readily appreciate that it ispreferred that the two half-antibodies used for the bispecific be thesame isotype.

Expression and Purification

Half-antibodies containing either the knob or hole mutations weregenerated in separate cultures by expressing the heavy and light chainsconstructs in a bacterial host cell, e.g., E. coli. The expressionplasmids were introduced into E. coli host strains 33D3 (Ridgway et al.(1999) 59 (11): 2718) or 64B4 (W3110 .DELTA.fhuA .DELTA.phoAilvG+.DELTA.prc spr43H1 .DELTA.degP .DELTA.manA lacI.sup.q .DELTA.ompT)and transformants were selected on carbenicillin containing LB plates.Transformants were then used to inoculate an LB starter culturecontaining carbenicillin, and this was grown overnight with shaking at30° C. The starter culture was diluted 100× into a phosphate limitingmedia C.R.A.P. (Simmons et al., 2002, J. Immunol. Methods, 263:133-147)containing carbenicillin, and the culture was grown for 24 hours withshaking at 30° C. The cultures were centrifuged, and the cell pelletsfrozen until the start of antibody purification. The pellets were thawedand resuspended in an extraction buffer containing 25 mM Tris-baseadjusted to pH 7.5 with hydrochloric acid, 125 mM NaCl and 5 mM EDTA(TEB or Tris Extraction Buffer) with a volume to weight ratio of 100 mLTEB per 5 grams of cell pellet, and extracted by disrupting the cellsusing microfluidics by passing the resuspended mixture through aMicrofluidics Corporation model 110F microfluidizer (Newton, Mass.)three times. The bacterial cell extract was then clarified bycentrifugation for 20 minutes at 15,000×g and the supernatant collectedand filtered through a 0.22 micron acetate filter prior to purification.

Each half-antibody was purified separately by Protein A affinitychromatography. Clarified cell extracts from the knob half-antibody wereloaded onto a 1 mL HiTrap MABSELECT SURE™ column from GE Healthcare(Pistcataway, N.J.) at 2 mL/min. After loading the column was washedwith 10 column volumes (CV) of 40 mM sodium citrate, pH 6.0, 125 mMsodium chloride, and 5 mM EDTA followed by 5 column volumes of 20 mMsodium citrate at pH 6.0 to facilitate capture by the cation exchangecolumn. The affinity captured half-antibodies were eluted with 10 columnvolumes (CV) of 0.2 mM acetic acid (pH 2-3).

Expression of Half-Antibodies in CHO Cells

Construction of Expression Plasmids.

Both heavy chain and light chain cDNAs were under the control ofCytomegalovirus immediate-early gene promoter and enhancer (CMV). EachCMV transcriptional start site is followed by splice donor and acceptorsequences, which define introns that are removed from the finaltranscripts (Lucas et al., High-level production of recombinant proteinsin CHO cells using a dicistronic DHFR intron expression vector. Nucl.Acid Res. (1996) 24:1774-9). The glutamine synthetase (GS) enzyme wasused as the selection marker for stable cell line development (Sanderset al., Amplification and cloning of the Chinese hamster glutaminesynthetase gene. The EMBO J. (1984) 3:65-71) and was under the controlof SV40 early promoter and enhancer.

Cell Culture.

CHO cells were cultured in a proprietary DMEM/F12-based medium in shakeflask vessels at 37° C. and 5% CO₂. Cells were passaged with a seedingdensity of 3×10⁵/mL, every three to four days.

Stable Transfection.

CHO cells were transfected using lipofectamine 2000 CD according to themanufacturer's recommendation (Invitrogen, Carlsbad, Calif.).Transfected cells were centrifuged and seeded into DMEM/F-12-basedselective (glutamine-free) medium with various concentrations ofmethionine sulfoximine (MSX). About three weeks after seeding,individual colonies were picked into 96-well plates. Picked colonieswere evaluated for antibody production by taking the supernatant forELISA analysis. Top clones were scaled-up and evaluated based onantibody titers, favorable metabolism (mainly lactate consumption), andacceptable product quality attributes.

Expression:

Each half antibody was expressed in CHO cells. 2 L cultures were grownand harvested.

Purification of Half-Antibodies.

Each half antibody was captured on a MABSELECT SURE™ column. The columnwas then washed with 4 column volumes (CV) of the following buffers: anequilibration buffer consisting of 50 mM TRIS pH 8.0, 150 mM NaCl, and awash buffer consisting of 0.4M Potassium Phosphate pH 7.0. Each arm waseluted into 0.15 M Sodium Acetate at pH 2.9.

The above described methods of expression and purification ofhalf-antibodies are generally applicable to IgG of different isotypes.

Example 2 Solubilizer & pH Hold

The following example details how the incubation of half-antibodies atan intermediate pH drove conformation shift and increased assemblyefficiency, and how the addition of a solubilizer such as arginine andhistidine reduced the intermediate pH-induced precipitation ofhalf-antibodies.

Half-antibody protein A pools are inherently unstable due to the exposedinner surface of the CH2 and CH3 domains which are likely to containhydrophobic patches which are normally in the non-solvent exposedsurface of an antibody. Thus, when adjusted to pH greater than 4half-antibody protein A pools tend to precipitate. The instant inventorsdiscovered that with a minimum concentration of L-Arginine present (incertain examples ≧50 mM) the half antibody was stabilized and remainedin solution upon pH adjustment. This addition of a solubilizer such asarginine kept the half antibody in solution, reduced turbidity upon pHadjustment, and increased bispecific assembly yield. See FIG. 3.Arginine also protected bispecific half antibodies and purifiedbispecific from forming aggregation during freezing. Similarprecipitation-reducing effect was also seen in histidine.

The recovered protein from the Protein A columns in Example 1 were usedas the starting material for this example.

L-Arginine (1M, pH 9) was added to the Protein A purified protein to afinal concentration of 50-600 mM. The solution was subsequently titratedto a higher pH using 1.5M Tris Base, pH 11, as needed. The step ofelevating to intermediate pH after acidic elution from the Protein Acolumn is referred to as intermediate pH hold.

Due to the knob and hole mutations in the CH3 domain bispecificantibodies have different degrees of flexibility compared to standardantibodies. As a result of this unique flexibility and the exposed innersurfaces of the CH2 and CH3 domains half antibodies appeared to undergoconformational shifts upon pH adjustment. See FIG. 2.

In this experiment, knobs underwent a shift from monomer tonon-covalently linked homodimer when the pH was adjusted to a pH greaterthan 4. See FIG. 2B. Holes underwent a conformation shift from a smallerhydrodynamic radius to a larger hydrodynamic radius based on SizeExclusion Chromatography retention time. The shift began to occur at pH5 and pH 7 drove the shift to completion. See FIG. 2A.

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel SW2000 column on an Agilent HPLC 1200 system. Protein (IgG1half-antibody produced in E. coli) was eluted with 0.2M K3PO4 0.25M KClpH 6.2 at a flow rate of 0.5 mL/min. The eluted protein was quantifiedby UV absorbance and integration of peak areas. See FIGS. 2 A & B. Thisshift may represent a folding intermediate that was induced by pH changedue to the higher flexibility of the half antibody, especially knob andhole half-antibodies with mutations in the CH3 domain.

The intermediate pH hold, however, may result in precipitation ofhalf-antibodies. As shown in FIG. 3A, the presence of arginine reducedpH-induced turbidity of the Protein A purified IgG1 knobhalf-antibodies. In this experiment, 1M arginine was used to titrate thepH in the E. coli produced-IgG1 half-antibody Protein A pools. The finalarginine concentration was about 50 mM when the pH was titrated to 5.5,about 200 mM at pH 7.5, and about 400 mM at pH 8.5 (see FIG. 3A). Thepresence of arginine also improved assembly yield of the knob-into-holebispecific antibody by 15% (data not shown).

Similarly, histidine was able to reduce pH-induced turbidity due toprecipitation. A “knob” IgG1 half-antibody was purified from E. colihomogenate on a MABSELECT SURE™ column, resulting in a Protein A poolwith a concentration of 12 g/L half-antibody. One-fourth volume ofArginine-Hydrochloride or Histidine-Hydrochloride was added to a finaladditive concentration of 200 mM, with an equivalent volume of purifiedwater added for the “None” control. Sample pH was increased usingconcentrated Sodium Hydroxide (50% w/v NaOH solution, or 19.1 N) addeddropwise, and data points were recorded. The pH was measured using anOrion Ross 81-03 microprobe. Turbidity of solution was measured using aHach 2100 laboratory turbidity meter.

The data in FIG. 3B showed that both arginine (200 mM) and histidine(200 mM) reduced pH-induced precipitation in IgG1 isolated from E. coli.In summary, intermediate pH induced half-antibody conformation shift infavor of bi-specific assembly, and a solubilizer added to theintermediate pH hold step reduced pH-induced precipitation.

Example 3 Reduction

The following example details how the use of a reducing conditiondecreases aggregation resulting in more formation of the desiredheteromultimer, e.g., a bispecific antibody. For example, glutathioneadded to an assembly mixture creates a weakly reducing condition that isadvantageous for knob-into-hole bispecific assembly. Other reductants ina similar class such as BMEA (Beta-MercaptoEthylAmine) may have asimilar effect.

Aggregation can occur during assembly of the knob and hole halfantibodies to form bispecific. Increasing glutathione levels minimizethe amount of aggregation during assembly. In contrast, strongreductants such as DTT at high concentrations may sometimes increaseaggregation. Without being limited to specific mechanisms, instead ofreducing the disulfide bonds permanently glutathione seems to shuffledisulfides acting as a catalyst for proper disulfide formation. Withglutathione assemblies a buffer exchange is not required in order toform the hinge region disulfides in the bispecific product of interest,as is required for reoxidation when using a strong reductant. Additionof a chemical re-oxidant is not required either when a weak reductantsuch as glutathione is used.

Glutathione concentrations can be expressed in terms of molarity or interms of molar ratio with respect to the amount of the hinge-containingpolypeptides or half-antibodies present in the assembly mixture. Using atarget molar ratio of reductant controls for the protein concentrationin the assembly mixture; this prevents over reducing or under reducingas a result of variable protein concentrations.

In this example, glutathione was added to the mixed half-antibodies from2 to 200× molar excess. The samples were incubated at room temperaturefor 46 hours. In the RP-HPLC (reversed phase-high performance liquidchromatography) all samples were diluted with 0.1% Trifluoroacetic Acidto a maximum concentration of 1.0 mg/ml. The protein concentration wasdetermined by photometric measurements at 280 nm. Four samples of 0.1%Trifluoroacetic Acid were injected prior to the sample analysis. Thisensured that the column was completely equilibrated. Protein A purifiedE coli-produced IgG1 half-antibodies were applied to a Poros R2/20 2.1mmD×20 mL on an Agilent HPLC 1200 system. Protein was eluted with alinear gradient of 38-49% Buffer A to 0.09% Trifluoroacetic Acid 80%Acetonitrile (Buffer B) in 20 minutes at a flow rate of 0.5 mL/min. Theeluted protein was quantified by UV absorbance and integration of peakareas. As shown in FIG. 4A, the level of bispecific formation increasedwith increased glutathione:Ab molar ratio. See FIG. 4A.

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed. Briefly,Protein A purified E coli-produced IgG1 half-antibodies were applied toa Tosoh TSKgel SW2000 column on an Agilent HPLC 1200 system. Protein waseluted with 0.2M K3PO4 0.25M KCl pH 6.2 at a flow rate of 0.5 mL/min.The eluted protein was quantified by UV absorbance and integration ofpeak areas. The 150 kD peak observed was confirmed to be due to theformation of bispecific antibody. See FIG. 4B.

As can be seen there is a shift in peaks from the unwanted monomers(i.e., half-antibodies, either knob or hole) and homodimers to theheteromultimer, i.e., a bispecific antibody (FIGS. 4A and B). Insummary, the data show that increasing molar ratio of glutathione tohalf-antibodies reduced aggregation and improved bispecific formation(FIG. 4B).

Example 4 Temperature

This example illustrates the effect of temperature on the stability ofhalf-antibodies and the assembly of the heteromultimer.

The temperature of the solution of the half-antibodies had a dramaticimpact on the rate of assembly. One example of enhanced assembly of E.coli-produced IgG1 half-antibodies at higher temperature is shown inFIG. 5A.

Another example showing the effect of temperature on bispecific assemblyis shown in FIG. 5B. In this experiment, two IgG1 half-antibodies wereproduced in E. coli and purified over Protein A as described inExample 1. The half antibodies were combined and divided into fouraliquots for testing bispecific assembly under different conditions withor without heating and/or intermediate pH hold.

As shown in FIG. 5B, a 200 molar excess of glutathione under varyingconditions enhanced the rate of bispecific IgG1 antibody formation. Thecontrol conditions (room temperature, about 20° C., half-antibody waskept at pH 4, no intermediate pH hold), allowed assembly of thebispecific albeit at a slower rate. Holding the Protein A purifiedhalf-antibodies at an intermediate pH (pH 5 for knob half-antibody; pH 7for hole half-antibody) for 16 hours at room temperature improved therate of bispecific antibody formation without going to a highertemperature (assembly mixture was at pH 8.5 in “pH optimized” in FIG.5B). Increasing temperature to 37° C. without an intermediate pH holdingstep increased the rate of bispecific antibody assembly over the controland it was faster relative to a pH holding step and the assembly done atroom temperature. The fastest assembly rate, however, was seen when theProtein A purified half-antibodies were held at an intermediate pH (asabove), then assembled at pH 8.5 at elevated temperature (i.e., 37° C.).Under this condition, about 80% of bispecific assembly was achieved inonly about 6 hours (FIG. 5B). In summary, the overall assembly rate wasincreased by heating, and the pH hold and heating had a synergisticeffect on assembly.

Heat-enhanced assembly was also seen in IgG4 bispecific antibody. Theresults of FIG. 6B show that in the presence of PVP and histidine,heated IgG4 half-antibodies produced by E. coli culture reached similarassembly results as the assembly of E. coli-produced IgG1half-antibodies. The amount of bispecific was analyzed using reversephase HPLC as described above. Taken together, the data show thatheating facilitated bi-specific formation.

Example 5 Stabilizers

The following example details how stabilizers can reduce aggregation asa result of heating and/or elevated pH during assembly and/orintermediate pH hold.

Polyvinylpyrrolidone (PVP) is a water soluble uncharged polymer with apyrrolidone group. PVP reduced aggregation during heated assembly.Without being limited to specific mechanisms, PVP can act to stabilize afolding intermediate of the bispecific or protect the half antibodiesfrom aggregation likely by interacting with the hydrophobic patches ofthe bispecific.

The effect of PVP on aggregate formation was analyzed using SEC underthe conditions as described in Example 3. Adding PVP minimized the highmolecular weight species (HMWS) present in the assembled pool to 12%HMWS with 4% PVP (w/v) compared to 4% NMWS without adding PVP. See FIG.6A. All samples were E. coli-produced IgG1 heated in the presence of 200mM arginine.

Next, the assembly of IgG4 bispecific antibody was tested. As shown inFIG. 6B, heated assembly in the presence of PVP and histidine greatlyimproved E. coli-produced IgG4 bispecific assembly to levels similar tothe heated assembly of IgG1 produced by E. coli shown in FIG. 5A.Arginine was present in both the heated sample and the room temperaturesample as a solubilizer and a pH titrant. In addition to PVP, anotherstabilizer Histidine was added before the heated intermediate pH holdstep to stabilize half-antibody during this step. The results show thatboth PVP and histidine improved IgG4 bispecific assembly to levelssimilar to IgG1 bispecific assembly (compare FIG. 6B with 5B).

FIG. 6C presents another example in which PVP minimized the formation ofHMWS during heated assembly of an IgG4 bispecific antibody.

Heating the Half-antibody Protein A pools accelerated the conformationshifts of the half antibodies upon incubation at an intermediate pH. Butheating can cause aggregation especially for IgG4 knob and holebispecific antibodies assembly from IgG4 half-antibodies produced in E.coli, Thus, additional solubilizer and/or stabilizers were added whenIgG4 half-antibodies were heated during assembly.

Conformation shift of E. coli IgG4 hole half-antibody was detected after48 hours of incubation at room temperature. When the IgG4 holehalf-antibodies were incubated at 37° C., conformation shift wasdetected in about three hours (data not shown). Heating, however, led toincrease in aggregation as determined by SEC. See FIG. 7, left panel. Inthis experiment, histidine was added during the heated intermediate pHhold step to test its effect on reducing aggregation of half-antibodies.As shown in FIG. 7, the presence of histidine during heating minimizedthe level of aggregation from 11% high molecular weight species (HMWS,no histidine) to 6% HMWS (200 mM histidine), without affectingconformation shift of the half antibodies. The results thus show that astabilizer reduced aggregate formation during both assembly ofbi-specific antibodies as well as intermediate pH hold ofhalf-antibodies.

Example 6 Assembly

This example provides protocols for two exemplary immunoglobulinisotypes, IgG1 and IgG4. The half-antibodies were produced in one of twodifferent host cells, i.e., either E. coli or CHO. It is understood thatthe methods described herein can be applied to other antibody isotypesproduced in the same or other sources. It is within the ability of oneskilled in the art to modify the protocols by routine experimentationbased on the knowledge in the art and the teachings disclosed in theinstant application.

Further, it is understood that formation of an antibody comprising ahalf-antibody produced in a first host cell (e.g., CHO) may be assembledwith a complementary half-antibody produced in a second host cell (e.g.,E. coli) (data not shown). Thus, for example, a knob half-antibodyproduced in CHO may be assembled with a hole half-antibody produced inE. coli or vice versa.

All four of the assembly procedures described in this example resultedin assemblies that plateaued after 4 hours and produced less than 10%aggregate and minimal aggregation during assembly.

A. IgG1 from E. coli:

Conformation Change of Half-Antibodies:

If the protein A pools were at a pH less than 7, the pH of bothhalf-antibody pools was adjusted to pH 7 using 1M Arginine (pH 9), andboth pools incubated at 37° C. for 3 hours. Alternatively, the poolswere incubated at room temperature for 48 hours. If the pools werealready at pH 7 for 48 hours or more, skip this step. The amount ofArginine added to get the solution to pH 7 was quantified.

The (still-warm) pools were combined, and the pH adjusted to pH 8.5using 1M Arginine (pH 9). The amount of Arginine added at this stage wasquantified.

2M Arginine (pH 8.5) was added until final concentration of Arginine was0.5 M.

200 mM reduced glutathione (GSH) in 0.5 M Arginine (final pH 8.5) wasadded until the GSH:Ab ratio was 200× (ex: add 6.88 μL of theglutathione solution for each mg of half-antibody).

The glutathione half-antibody solution was incubated at 37° C. for 4hours to allow the half-antibodies to assemble into a bispecificantibody.

B. IgG1 from CHO:

Assembled as described above for E. coli. The pH may not need to beraised to pH 7 or above for the intermediate pH hold. Assembly timeafter the addition of glutathione may reach completion after 2 hours.

C. IgG4 from CHO:

Assembled under the same conditions as above (IgG1 from CHO). Histidineand PVP may not be required.

D. IgG4 from E. coli:

Assembling IgG4 from E. coli using the above protocols resulted in high,i.e., ˜35%, aggregate levels. This necessitated modifications ofassembly conditions:

It was determined that the Protein A pool composition containing 0.2 Mhistidine and 50 mM Arginine yielded acceptable results (data notshown). Thus, several ways to provide the end result were investigatedand determined to provide acceptable results.

A first method used altered elution (pH 3) and wash buffers (pH 7)during Protein A purification to contain 0.2 M His & 50 mM Arg. Thisresulted in the final Protein A pool containing 0.2M His and 50 mM Arg,pH 4, that was then titrated to pH 8 using 1.5M Tris base (pH 11).

A second alternative method utilized a 0.8M solution of Histidine HCl(which has a solubility of 0.8 M). A one third-volume of the HistidineHCl was added to the Protein A pool(s) to reach a final concentration of0.2M His. Then a 1/40th volume of 2M Arg was added.

A third alternative method was to Buffer-exchange the Protein A poolsinto a 0.2 M His & 50 mM Arg buffer (preferably at pH 8).

A final alternative method was to add Histidine directly to the ProteinA pool(s) (31.03 g/L), then add 1/40th volume of 2M Arg.

A one quarter-volume of a 20% w/v solution of Spectrum PVP K-15 in 0.2MHistidine and 50 mM Arg was added to the Protein A pool(s).

It was noted that PVP also minimized aggregation during assembly forIgG1 under low glutathione conditions.

Conformation change of half-antibodies: The pH of the protein A pool(s)was adjusted to pH 8.0 using 1.5M Tris Base with 0.2M Histidine and 50mM Arg and 4% PVP K-15 (pH 11) and incubated at 37° C. for 3 hours.Alternately, the pool(s) could be incubated at room temperature for 48hours.

200 mM reduced glutathione (GSH) in 0.2 M Histidine, 4% PVP, 50 mMArginine; final pH 8.0, was added until the GSH:Ab ratio was 200× (ex:add 6.88 μL of the glutathione solution for each mg of half-antibody).If the half-antibody pools had not been combined they were combined atthis point.

The pooled half-antibodies were incubated at 37° C. for 4 hours to allowfor the formation of the bispecific antibody. At this point, the percentbispecific antibody has plateaued.

Once the amount of bispecific antibody has plateaued the solution can bestored at low temperature or adjusted to a lower pH for processing onsubsequent chromatography steps.

The methods disclosed herein find use in the manufacture of therapeuticproteins such as bispecific antibodies.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of producing a heteromultimeric proteincomprising the steps of: a. providing a first half-antibody at pH 5-9 inthe presence of a first solubilizer, wherein the first half-antibodycomprises a heteromultimerization domain; b. providing a secondhalf-antibody at pH 5-9 in the presence of a second solubilizer, whereinthe second half-antibody comprises a heteromultimerization domain; c.mixing the first and second half-antibodies to form an assembly mixturein a reducing condition comprising 50-400× molar excess of glutathione(GSH) with respect to the total amount of half-antibodies; and d.incubating the assembly mixture to produce a heteromultimeric proteincomprising the first and second half-antibodies, wherein the firsthalf-antibody interacts with the second half-antibody at theheteromultimerization domain.
 2. The method of claim 1, wherein thefirst solubilizer or the second solubilizer is selected from the groupconsisting of arginine, histidine and sucrose.
 3. The method of claim 1,wherein the first solubilizer or the second solubilizer is arginine, anarginine salt or arginine derivative.
 4. The method of claim 1, whereinthe first solubilizer or the second solubilizer is histidine, ahistidine salt or histidine derivative.
 5. The method of claim 2,wherein the first solubilizer or the second solubilizer is present at aconcentration of between 20 mM and 1M.
 6. The method of claim 5, whereinthe first solubilizer or the second solubilizer is present at aconcentration of between 20 mM and 400 mM.
 7. The method of claim 1wherein the first and second solubilizers are arginine HCl.
 8. Themethod of claim 1, wherein the first half-antibody and the secondhalf-antibody are purified before mixing.
 9. The method of claim 1,wherein the first half-antibody and the second half-antibody areco-purified.
 10. The method of claim 1, wherein the step a is precededby the step of purifying the first half-antibody or the step b ispreceded by the step of purifying the second half-antibody.
 11. Themethod of claim 10, wherein the first half-antibody and the secondhalf-antibody are purified by protein A.
 12. The method of claim 1,wherein the first and second half-antibodies are produced by a bacterialcell, a yeast cell, a baculovirus, or a mammalian cell.
 13. The methodof claim 12, wherein the first and second half-antibodies are producedby a mammalian cell.
 14. The method of claim 13, wherein the mammaliancell is a CHO cell.
 15. The method of claim 1, wherein the first orsecond half-antibody is an IgG half-antibody.
 16. The method of claim15, wherein the IgG half-antibody is of the IgG1, IgG2 or IgG4 isotype.17. The method of claim 15, wherein the first or second half-antibodycomprises an Fc component.
 18. The method of claim 15, wherein the firstor second half-antibody comprises a V_(L) domain, a V_(H) domain, ahinge domain, a CH₂ domain and a CH₃ domain.
 19. The method of claim 18,wherein the first or second half-antibody comprises a single chainpolypeptide that further comprises a tether, and wherein said singlechain polypeptide comprises domains positioned relative to each other inan N-terminal to C-terminal direction as follows:V_(L)-tether-V_(H)-hinge-CH₂—CH₃.
 20. The method of claim 18, whereinthe first or second half-antibody further comprises a C_(L) domain and aCH₁ domain.
 21. The method of claim 20, wherein the first or secondhalf-antibody comprises a single chain polypeptide that furthercomprises a tether, and wherein said single chain polypeptide comprisesdomains positioned relative to each other in an N-terminal to C-terminaldirection as follows: V_(L)—C_(L)-tether-V_(H)—CH₁-hinge-CH₂—CH₃. 22.The method of claim 1, wherein one or more of steps a-d are heated at atemperature of between 25° C. and 42° C.
 23. The method of claim 22,wherein one or more of steps a-d are heated at a temperature of between32° C. and 37° C.
 24. The method of claim 22, wherein the firsthalf-antibody of step a and the second half-antibody of step b areheated.
 25. The method of claim 22, wherein the assembly mixture of stepd is heated.
 26. The method of claim 22, wherein all of steps a-d areheated.
 27. The method of claim 26, wherein all of steps a-d are heatedat a temperature between 32° C. and 37° C.
 28. The method of claim 1,wherein the reducing condition has an oxidation potential of between−200 to −600 mV.
 29. The method of claim 1, wherein the GSH is added in100-300× molar excess to the assembly mixture.
 30. The method of claim29, wherein the GSH is added in 200× molar excess to the assemblymixture.
 31. The method of claim 1 wherein the assembly mixture isincubated at pH 7 to pH
 9. 32. The method of claim 1, wherein theheteromultimerization domain of the first or second half antibodycomprises one or more of a knob, a hole, a leucine zipper, a coiledcoil, or a polar amino acid residue capable of forming an electrostaticinteraction.
 33. The method of claim 32, wherein theheteromultimerization domain of the first half-antibody comprises a knoband the heteromultimerization second domain of the half-antibodycomprises a hole.
 34. The method of claim 1, further comprising adding astabilizer in one or more of steps a-d.
 35. The method of claim 34,wherein the stabilizer is added to step c or step d.
 36. The method ofclaim 35, wherein the stabilizer is arginine or polyvinylpyrrolidone(PVP).
 37. The method of claim 1, further comprising the step ofrecovering the heteromultimeric protein.
 38. The method of claim 37,wherein the step of recovering the heteromultimeric protein comprisespurifying the heteromultimeric protein.
 39. A method of producing amultispecific antibody comprising the steps of a. providing a firsthalf-antibody at pH 5-9 in the presence of arginine or histidine,wherein the first half-antibody comprises a heteromultimerizationdomain; b. providing a second half-antibody at pH 5-9 in the presence ofarginine or histidine, wherein the second half-antibody comprises aheteromultimerization domain; c. mixing the first and secondhalf-antibodies to form an assembly mixture in a reducing conditioncomprising 50-400× molar excess of glutathione (GSH) with respect to thetotal amount of the half-antibodies; and d. incubating the assemblymixture to form a multispecific antibody comprising the first and secondhalf-antibodies, wherein the first half-antibody interacts with thesecond half-antibody at the heteromultimerization domain.
 40. The methodof claim 39, wherein the arginine or histidine is present at aconcentration of between 20 mM and 400 mM.
 41. The method of claim 39,wherein the first half-antibody and the second half-antibody arepurified before mixing.
 42. The method of claim 39, wherein the firstand second half-antibodies are produced by a bacterial cell, a yeastcell, a baculovirus, or a mammalian cell.
 43. The method of claim 42,wherein the first and second half-antibodies are produced by a mammaliancell.
 44. The method of claim 43, wherein the mammalian cell is a CHOcell.
 45. The method of claim 39, wherein the first or secondhalf-antibody is of the IgG1, IgG2 or IgG4 isotype.
 46. The method ofclaim 39, wherein the first or second half-antibody comprises an Fccomponent.
 47. The method of claim 39, wherein one or more of steps a-dare heated at a temperature of between 25° C. and 42° C.
 48. The methodof claim 39, wherein the GSH is added in 100-300× molar excess to theassembly mixture.
 49. The method claim 39, wherein theheteromultimerization domain of the first half antibody or the secondhalf antibody comprises one or more of a knob, a hole, a leucine zipper,a coiled coil, or a polar amino acid residue capable of forming anelectrostatic interaction.
 50. The method of claim 49, wherein theheteromultimerization domain of the first half-antibody comprises a knoband the heteromultimerization domain of the second half-antibodycomprises a hole.
 51. The method of claim 39, further comprising addinga stabilizer in one or more of steps a-d.
 52. The method of claim 51,wherein the stabilizer is added to step c or step d.
 53. The method ofclaim 52, wherein the stabilizer is arginine or polyvinylpyrrolidone(PVP).
 54. The method of claim 39, further comprising the step ofrecovering the multispecific antibody.
 55. The method of claim 54,wherein the step of recovering the multispecific antibody comprisespurifying the multispecific antibody.
 56. A method of producing aheteromultimeric protein, said method comprising: a. obtaining a proteinA purified first half-antibody, wherein the first half-antibodycomprises a heterodimerization domain; b. obtaining a protein A purifiedsecond half-antibody, wherein the second half-antibody comprises aheterodimerization domain; c. adjusting the pH of each half-antibodyhinge containing polypeptide to between pH 4 and 9; d. mixing the firstand second half-antibodies to obtain an assembly mixture, e. adding a50-400× molar excess of glutathione (GSH) to the assembly mixture; andf. incubating the assembly mixture to form a heteromultimeric proteincomprising the first half antibody and the second half-antibody.
 57. Themethod of claim 56, wherein the first and second half-antibody comprisesan Fc component.
 58. The method of claim 56, wherein in step c the pH ofthe first and second half-antibody is adjusted to pH 4-9 in the presenceof a solubilizer.
 59. The method of claim 58, wherein the solubilizer isarginine or histidine that is added to a final concentration of between20 mM and 1 M prior to adjusting the pH.
 60. The method of claim 56,wherein the heteromultimerization domain of the first or second halfantibody comprises one or more of a knob, a hole, leucine zippers, acoiled coil, or a polar amino acid residue capable of forming anelectrostatic interaction.
 61. The method of claim 60, wherein theheteromultimerization domain of the first half-antibody comprises a knoband the heteromultimerization domain of the second half-antibodycomprises a hole.
 62. The method of claim 56, wherein the pH is adjustedafter mixing.
 63. The method of claim 56, further comprising incubatingthe assembly mixture at a temperature of between 15° C. and 39° C. forat least 30 minutes.
 64. The method of claim 56, wherein the assemblymixture in step f has an oxidation potential of between −200 to −600 mV.65. The method of claim 56, wherein incubating the assembly mixture isdone at a temperature between 15° C. and 39° C. in the presence ofPolyvinylpyrrolidone (PVP).
 66. The method of claim 65, wherein the PVPis added up to 40% (w/v).
 67. The method of 56, wherein the first orsecond half-antibody is produced by a bacterial cell, a yeast cell, abaculovirus or a mammalian cell.
 68. The method of claim 67, wherein thefirst or second half-antibody is produced by a CHO cell.
 69. The methodof claim 56, wherein the GSH is added in 100-300× molar excess.
 70. Themethod of claim 56, wherein the GSH is added in 200× molar excess. 71.The method of claim 61, wherein the knob comprises a T366W substitution,and the hole comprises T366S, L368A, and Y407V substitutions.